Search Results (20)

Conus venoms are both highly powerful and complex, exhibiting a remarkably intriguing molecular variability. The biologic reasons behind such astonishing molecular diversity are yet to be fully understood. We hypothesized that the current knowledge has been hampered by a lack of studies targeting the whole Conus genus backed by a feeding habit analysis, as opposed to the abundant studies focused on single species or at the individual level. We aim to enlighten the understanding of the remarkable venom variability in cone snails while pushing to deliver novel peptides for biomedical applications through a broad transcriptomics approach. Here, we assessed 76 publicly available venom-related and unrelated transcriptomes from a total of 20 different Conus species. The shared transcriptomic repertoire revealed several gene variations in accordance with predatory diets (e.g., gene loss in piscivorous species), indicating that feeding habit largely influences venom evolution. Furthermore, evidences of ubiquitous symbiotic relationships within the venom organs were depicted, as biological processes alien to Conus species (e.g., Sorocarp morphogenesis) were found in all analyzed transcriptomes. Moreover, 88 potential anti-microbial peptides were bioinformatically detected, including one showing similarity with the human ACE2 receptor. Our study highlights the importance of in-depth comparative transcriptomic analyses, fostering cross-field synergic assessments by relying on informatic, biologic, and pharmacologic resources. Full article

Background: We previously showed that peptides encompassing the unique b3a2 or b2a2 breakpoint amino-acid sequence of oncogenic p210 induced peptide-specific T-cell responses in chronic myeloid leukemia (CML) patients. Methods: From 2007 to 2011, two multicenter peptide vaccine phase II studies, GIMEMA CML0206 and SI0207, enrolling overall 109 CML patients (68 b3a2 and 41 b2a2) with persistence of molecular disease during imatinib treatment, were carried out. Peptide vaccination schedule included the following: “immunization phase” (six vaccinations every 2 weeks); “reinforcement” phase (three monthly boosts) and “maintenance” phase (two boosts at 3-month intervals). GM-CSF (granulocyte-macrophage-colony-stimulating factor, sarmograstim) served as the immunological adjuvant. Results: The short-term results (at completion of vaccine protocol—12 months) and long-term follow-up are reported. All patients completed the vaccination schedule with no toxicity. After vaccinations, the BCR::ABL1 peptide-specific CD4+ T-cell response was documented in 80% of patients. In the short term, 30% of patients achieved a reduction in BCR::ABL1, while the majority showed stable molecular disease with fluctuations. The median follow-up since diagnosis and last vaccination are 18 and 10 years, respectively, with an overall survival (OS) rate at 18 years of 89%. In addition, 97/109 (89%) patients are alive, while 12/109 (11%) died of CML-unrelated reasons. Overall, 18/109 (16.5%) patients are in treatment-free remission (TFR) for a median time of 48 months. Conclusions: The long-term results of p210 peptide vaccinations in CML patients with persisting disease during imatinib treatment showed its feasibility, safety, absence of off-targets events, high OS and not negligible rate of successful TFR. Active immunotherapeutic approaches in CML patients with low disease burden, eventually employing newer vaccine strategies such as mRNA vaccines, may be reconsidered. Full article

B30.2 domains, sometimes referred to as PRY/SPRY domains, were originally identified by sequence profiling methods at the gene level. The B30.2 domain comprises a concanavalin A-like fold consisting of two twisted seven-stranded anti-parallel β-sheets. B30.2 domains are present in about 150 human and 700 eukaryotic proteins, usually fused to other domains. The B30.2 domain represents a scaffold, which, through six variable loops, binds different unrelated peptides or endogenous low-molecular-weight compounds. At the cellular level, B30.2 proteins engage in supramolecular assemblies with important signaling functions. In humans, B30.2 domains are often found in E3-ligases, such as tripartite motif (Trim) proteins, SPRY domain-containing SOCS box proteins, Ran binding protein 9 and −10, Ret-finger protein-like, and Ring-finger proteins. The B30.2 protein recognizes the target and recruits the E2-conjugase by means of the fused domains, often involving specific adaptor proteins. Further well-studied B30.2 proteins are the methyltransferase adaptor protein Ash2L, some butyrophilins, and Ryanodine Receptors. Although the affinity of an isolated B30.2 domain to its ligand might be weak, it can increase strongly due to avidity effects upon recognition of oligomeric targets or in the context of macromolecular machines. Full article

Emerging evidence suggests that glucagon-like peptide-1 receptor (GLP1R) agonists may have potential benefits for mental illnesses. However, their exact effects remain unclear. This study investigated the causal relationship between glucagon-like peptide-1 receptor agonist (GLP1RA) and the risk of 10 common mental illnesses, including attention deficit and hyperactivity disorder, anorexia nervosa, anxiety disorder, autism spectrum disorder, bipolar disorder, major depressive disorder, post-traumatic stress disorder, schizophrenia, cannabis use disorder, and alcohol use disorder. We selected GLP1RA as the exposure and conducted a Mendelian randomization (MR) analysis. The cis-eQTLs of the drug target gene GLP1R, provided by eQTLGen, were used to simulate the pharmacological effects of GLP1RA. Type 2 diabetes and BMI were included as positive controls. Using data from both the Psychiatric Genomic Consortium and FinnGen, we conducted separate MR analyses for the same disease across these two independent databases. Meta-analysis was used to pool the results. We found genetic evidence suggesting a causal relationship between GLP1RA and a reduced risk of schizophrenia [OR (95% CI) = 0.84 (0.71–0.98), I² = 0.0%, common effects model]. Further mediation analysis indicated that this effect might be unrelated to improvements in glycemic control but rather mediated by BMI. However, the findings of this study provide insufficient evidence to support a causal relationship between GLP1RA and other mental illnesses. Sensitivity analyses did not reveal any potential bias due to horizontal pleiotropy or heterogeneity in the above results (p > 0.05). This study suggests that genetically proxied activation of glucagon-like peptide-1 receptor is associated with a lower risk of schizophrenia. GLP1R is implicated in schizophrenia pathogenesis, and its agonists may exert potential benefits through weight management. Our study provides useful information for understanding the neuropsychiatric effects of GLP1RA, which may contribute to refining future research designs and guiding clinical management. Moreover, our findings could have significant implications for overweight individuals at high risk of schizophrenia when selecting weight-loss medications. Future research should further investigate the potential mechanisms underlying the relationship between GLP1RA and schizophrenia. Full article

Phage display has been widely used to identify peptides binding to a variety of biological targets. In the current work, we planned to select novel peptides targeting CD4 through screening of a commercial phage display library (New England Biolabs Ph.D.^TM-7). After three rounds of biopanning, 57 phage clones were Sanger-sequenced. These clones represented 30 unique peptide sequences, which were subjected to phage ELISA, resulting in the identification of two potential target binders. Following peptide synthesis, downstream characterization was conducted using fluorescence plate-based assay, flow cytometry, SPR, and confocal microscopy. The results revealed that neither of the peptides identified in the Sanger-based phage display selection exhibited specific binding toward CD4. The naïve library and the phage pool recovered from the third round of biopanning were then subjected to next-generation sequencing (NGS). The results of NGS indicated corruption of the selection output by a phage already known as a fast-propagating clone whose target-unrelated enrichment can shed light on the misidentification of target-binding peptides through phage display. This work provides an in-depth insight into some of the challenges encountered in peptide phage display selection. Furthermore, our data highlight that NGS, by exploring a broader sequence space and providing a more precise picture of the composition of biopanning output, can be used to refine the selection protocol and avoid misleading the process of ligand identification. We hope that these findings can describe some of the complexities of phage display selection and offer help to fellow researchers who have faced similar situations. Full article

Enhancing cross-protections against diverse influenza viruses is desired for influenza vaccinations. Neuraminidase (NA)-specific antibody responses have been found to independently correlate with a broader influenza protection spectrum. Here, we report a sequential immunization regimen that includes priming with NA protein followed by boosting with peptide nanoclusters, with which targeted enhancement of antibody responses in BALB/c mice to certain cross-protective B-cell epitopes of NA was achieved. The nanoclusters were fabricated via desolvation with absolute ethanol and were only composed of composite peptides. Unlike KLH conjugates, peptide nanoclusters would not induce influenza-unrelated immunity. We found that the incorporation of a hemagglutinin peptide of H2-d class II restriction into the composite peptides could be beneficial in enhancing the NA peptide-specific antibody response. Of note, boosters with N2 peptide nanoclusters induced stronger serum cross-reactivities to heterologous N2 and even heterosubtypic N7 and N9 than triple immunizations with the prototype recombinant tetrameric (rt) N2. The mouse challenge experiments with HK68 H3N2 also demonstrated the strong effectiveness of the peptide nanocluster boosters in conferring heterologous protection. Full article

Calcitonin gene-related peptide (CGRP) antagonists are a class of medications that act as antagonists of the CGRP receptor or ligand. They can be divided into monoclonal antibodies and non-peptide small molecules, also known as gepants. CGRP antagonists were the first oral agents specifically designed to prevent migraines. The second generation of gepants includes rimegepant (BHV-3000, BMS-927711), ubrogepant (MK-1602), and atogepant (AGN-241689, MK-8031). Zavegepant (BHV-3500, BMS-742413) belongs to the third generation of gepants characterized by different administration routes. The chemical and pharmacological properties of this new generation of gepants were calculated. The clinical trials showed that the new generation of CGRP antagonists is effective for the acute and/or preventive treatment of migraines. No increased mortality risks were observed to be associated with the second- and third-generation gepants. Moreover, the majority of the serious adverse events reported probably occurred unrelated to the medications. Interesting facts about gepants were highlighted, such as potency, hepatotoxicity, concomitant use with monoclonal antibodies targeting the CGRP, comparative analysis with triptans, and the “acute and preventive” treatment of migraine. Further studies should include an elderly population and compare the medications inside this class and with triptans. There are still concerns regarding the long-term side effects of these medications, such as chronic vascular hemodynamic impairment. Meanwhile, careful pharmacovigilance and safety monitoring should be performed in the clinical practice use of gepants. Full article

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage. Full article

The nonspecific enrichment of target-unrelated peptides during biopanning remains a major drawback for phage display technology. The commercial Ph.D.^TM-7 phage display library is used extensively for peptide discovery. This library is based on the M13KE vector, which carries the lacZα sequence, leading to the formation of blue plaques on IPTG-X-gal agar plates. In the current study, we report the isolation of a fast-propagating white clone (displaying WSLGYTG peptide) identified through screening against a recombinant protein. Sanger sequencing demonstrated that white plaques are not contamination from environmental M13-like phages, but derive from the library itself. Whole genome sequencing revealed that the white color of the plaques results from a large 827-nucleotide genomic deletion. The phenotypic characterization of propagation capacity through plaque count- and NGS-based competitive propagation assay supported the higher propagation rate of Ph-WSLGYTG clone compared with the library. According to our data, white plaques are likely to arise endogenously in Ph.D. libraries due to mutations in the M13KE genome and should not always be viewed as exogenous contamination. Our findings also led to the conclusion that the deletion observed here might be an ancestral mutation already present in the naïve library, which causes target-unrelated nonspecific enrichment of white clone during biopanning due to propagation advantage. Full article

Presenilin 2 (PS2), one of the three proteins in which mutations are linked to familial Alzheimer’s disease (FAD), exerts different functions within the cell independently of being part of the γ-secretase complex, thus unrelated to toxic amyloid peptide formation. In particular, its enrichment in endoplasmic reticulum (ER) membrane domains close to mitochondria (i.e., mitochondria-associated membranes, MAM) enables PS2 to modulate multiple processes taking place on these signaling hubs, such as Ca²⁺ handling and lipid synthesis. Importantly, upregulated MAM function appears to be critical in AD pathogenesis. We previously showed that FAD-PS2 mutants reinforce ER–mitochondria tethering, by interfering with the activity of mitofusin 2, favoring their Ca²⁺ crosstalk. Here, we deepened the molecular mechanism underlying PS2 activity on ER–mitochondria tethering, identifying its protein loop as an essential domain to mediate the reinforced ER–mitochondria connection in FAD-PS2 models. Moreover, we introduced a novel tool, the PS2 loop domain targeted to the outer mitochondrial membrane, Mit-PS2-LOOP, that is able to counteract the activity of FAD-PS2 on organelle tethering, which possibly helps in recovering the FAD-PS2-associated cellular alterations linked to an increased organelle coupling. Full article

CD8⁺ T-cell responses to pathogens are directed against infected cells that present pathogen-encoded peptides on MHC class-I molecules. Although natural responses are polyclonal, the spectrum of peptides that qualify for epitopes is remarkably small even for pathogens with high coding capacity. Among those few that are successful at all, a hierarchy exists in the magnitude of the response that they elicit in terms of numbers of CD8⁺ T cells generated. This led to a classification into immunodominant and non-immunodominant or subordinate epitopes, IDEs and non-IDEs, respectively. IDEs are favored in the design of vaccines and are chosen for CD8⁺ T-cell immunotherapy. Using murine cytomegalovirus as a model, we provide evidence to conclude that epitope hierarchy reflects competition on the level of antigen recognition. Notably, high-avidity cells specific for non-IDEs were found to expand only when IDEs were deleted. This may be a host’s back-up strategy to avoid viral immune escape through antigenic drift caused by IDE mutations. Importantly, our results are relevant for the design of vaccines based on cytomegaloviruses as vectors to generate high-avidity CD8⁺ T-cell memory specific for unrelated pathogens or tumors. We propose the deletion of vector-encoded IDEs to avoid the suppression of epitopes of the vaccine target. Full article

Next generation sequencing (NGS) in combination with phage surface display (PSD) are powerful tools in the newly equipped molecular biology toolbox for the identification of specific target binding biomolecules. Application of PSD led to the discovery of manifold ligands in clinical and material research. However, limitations of traditional phage display hinder the identification process. Growth-based library biases and target-unrelated peptides often result in the dominance of parasitic sequences and the collapse of library diversity. This study describes the effective enrichment of specific peptide motifs potentially binding to arsenic as proof-of-concept using the combination of PSD and NGS. Arsenic is an environmental toxin, which is applied in various semiconductors as gallium arsenide and selective recovery of this element is crucial for recycling and remediation. The development of biomolecules as specific arsenic-binding sorbents is a new approach for its recovery. Usage of NGS for all biopanning fractions allowed for evaluation of motif enrichment, in-depth insight into the selection process and the discrimination of biopanning artefacts, e.g., the amplification-induced library-wide reduction in hydrophobic amino acid proportion. Application of bioinformatics tools led to the identification of an SxHS and a carboxy-terminal QxQ motif, which are potentially involved in the binding of arsenic. To the best of our knowledge, this is the first report of PSD combined with NGS of all relevant biopanning fractions. Full article

Venoms act with remarkable specificity upon a broad diversity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake (Pseudechis porphyriacus) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease. Full article

Intercellular gap junction (GJ) contacts formed by the coupling of connexin (Cx) hemichannels (HCs) embedded into the plasma membranes of neighboring cells play significant role in the development, signaling and malfunctions of mammalian tissues. Understanding and targeting GJ functions, however, calls for finding valid Cx subtype-specific inhibitors. We conjecture the lack of information about binding interactions between the GJ interface forming extracellular EL1 and EL2 loops and peptide mimetics designed to specifically inhibit Cx43HC coupling to Cx43GJ. Here, we explore active spots at the GJ interface using known peptide inhibitors that mimic various segments of EL1 and EL2. Binding interactions of these peptide inhibitors and the non-peptide inhibitor quinine has been modelled in combination with the use of blind docking molecular mechanics (MM). The neuron-specific Cx36HC and astrocyte-specific Cx43HC subtypes were modelled with a template derived from the high-resolution structure of Cx26GJ. GJ-coupled and free Cx36HC and Cx43HC models were obtained by dissection of GJs (GJ-coupled) followed by 50 ns molecular dynamics (free). Molecular mechanics (MM) calculations were performed by the docking of inhibitors, explicitly the designed Cx43 EL1 or EL2 loop sequence mimetics (GAP26, P5 or P180–195, GAP27, Peptide5, respectively) and the Cx36 subtype-specific quinine into the model structures. In order to explore specific binding interactions between inhibitors and CxHC subtypes, MM/Generalized Born Surface Area (MM/GBSA) ΔG_bind values for representative conformers of peptide mimetics and quinine were evaluated by mapping the binding surface of Cx36HC and Cx43HC for all inhibitors. Quinine specifically contacts Cx36 EL1 residues V54-C55-N56-T57-L58, P60 and N63. Blocking the vestibule by the side of Cx36HC entry, quinine explicitly interacts with the non-conserved V54, L58, N63 residues of Cx36 EL1. In addition, our work challenges the predicted specificity of peptide mimetics, showing that the docking site of peptides is unrelated to the location of the sequence they mimic. Binding features, such as unaffected EL2 residues and the lack of Cx43 subtype-specificity of peptide mimetics, suggest critical roles for peptide stringency and dimension, possibly pertaining to the Cx subtype-specificity of peptide inhibitors. Full article

Therapeutic vaccinations aim to re-educate human immunodeficiency virus (HIV)-1-specific immune responses to achieve durable control of HIV-1 replication in virally suppressed infected individuals after antiretroviral therapy (ART) is interrupted. In a double blinded, placebo-controlled phase IIa multicenter study, we investigated the safety and immunogenicity of intranodal administration of the HIVACAT T cell Immunogen (HTI)-TriMix vaccine. It consists of naked mRNA based on cytotoxic T lymphocyte (CTL) targets of subdominant and conserved HIV-1 regions (HTI), in combination with mRNAs encoding constitutively active TLR4, the ligand for CD40 and CD70 as adjuvants (TriMix). We recruited HIV-1-infected individuals under stable ART. Study-arms HTI-TriMix, TriMix or Water for Injection were assigned in an 8:3:3 ratio. Participants received three vaccinations at weeks 0, 2, and 4 in an inguinal lymph node. Two weeks after the last vaccination, immunogenicity was evaluated using ELISpot assay. ART was interrupted at week 6 to study the effect of the vaccine on viral rebound. The vaccine was considered safe and well tolerated. Eighteen percent (n = 37) of the AEs were considered definitely related to the study product (grade 1 or 2). Three SAEs occurred: two were unrelated to the study product, and one was possibly related to ART interruption (ATI). ELISpot assays to detect T cell responses using peptides covering the HTI sequence showed no significant differences in immunogenicity between groups. There were no significant differences in viral load rebound dynamics after ATI between groups. The vaccine was safe and well tolerated. We were not able to demonstrate immunogenic effects of the vaccine. Full article