Search Results (487)

Budding yeast Tiny Yeast Comet 1 (Tyc1) was previously identified based on homology to human p31^comet. The conserved homologous region is within the previously mapped p31^comet Taxol-stabilized microtubule binding domain at the C-terminus of p31^comet. The p31^comet protein mimics the 3-dimensional shape of the HORMA-domain protein Mitotic Arrest-Deficient 2 (Mad2). Several HORMA-domain proteins have been reported to form polymers. By employing negative staining electron microscopy, we observed that the Tyc1 protein forms comet-tail-shaped polymers in association with Taxol-stabilized microtubules. When associated with microtubules, Tyc1p polymers frequently displayed a braid-like appearance around and off the ends of the microtubules. Analysis of two mutant forms of Tyc1p revealed that the amino acid motifs that are conserved between Tyc1p and human p31^comet were essential for robust polymer formation. Tyc1p polymers that were formed in the presence of Mad2p in association with a Mad2-binding motif peptide were able to suppress Taxol-stabilized microtubule depolymerization that was induced by exposure to ice-cold CaCl₂. In conclusion, yeast Tyc1p forms polymers that can suppress Taxol-stabilized microtubule depolymerization, potentially yielding an insight into a microtubule-associated function for human p31^comet. Full article

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are conserved microbial proteins that contain immunogenic patterns capable of activating plant pattern-triggered immunity (PTI). NLP patterns from Sclerotinia sclerotiorum (SsNLPs), a destructive necrotrophic fungal pathogen with a broad host range, have been identified, and their roles in PTI have been revealed. Nevertheless, the molecular mechanisms by which SsNLPs stimulate plant immunity remain largely unknown. In this study, we phylogenetically characterized SsNLPs and demonstrated the involvement of the phytocytokine receptor-like kinases PEPRs in SsNLP1-triggered immunity. SsNLPs contained the NPP1 domain and GHRHDWE motif and were phylogenetically closely associated with Botrytis cinerea NLPs. SsNLP1 treatment strongly induced the expression of PEPR genes. Further genetic analyses using Arabidopsis wild-type, pepr1 pepr2 double mutant, and PEPR1 overexpression lines showed that SsNLP1 elicited diverse immune responses, including reactive oxygen species (ROS) accumulation and defense gene activation, and induced plant resistance to S. sclerotiorum. Notably, the induced plant resistance and immune responses were strengthened in PEPR1 overexpression lines and significantly reduced in the pepr1 pepr2 mutant, indicating a positive role of PEPR signaling in SsNLP1-triggered immunity. Overall, our results revealed that phytocytokine PEPR1 signaling amplifies PAMP SsNLP1-triggered immunity, thereby enhancing resistance against S. sclerotiorum. Our findings provide an example of the coordination between PAMP- and phytocytokine-triggered immunity for robust resistance to a necrotrophic pathogen. Full article

The Lysobacter capsici XL1 β-lytic protease (Blp) is a bacteriolytic enzyme that hydrolyzes peptide bonds in the interpeptide bridge of the peptidoglycan of Gram-positive bacteria, including antibiotic-resistant strains of pathogenic bacteria. The Blp has been extensively characterized. The only unexplored aspect is the mechanism by which this enzyme recognizes target cells. In this work, we demonstrated for the first time that the Blp structure contained a C-terminal subdomain that can be responsible for this interaction. Molecular modeling suggested a hydrophobic nature of the interaction between the Blp and peptidoglycan. Model mutant forms of the Blp, which have fewer hydrophobic areas in the C-terminal subdomain, also had fewer sites for potential interaction with the ligand. Wet lab experiments showed that these mutant Blp forms exhibited poorer binding to peptidoglycan and living Staphylococcus aureus 209P cells, resulting in decreased bacteriolytic and proteolytic activity. Amino acid residues N136 and Y160 in the C-terminal subdomain were identified and can be important for the interaction of the enzyme with target cells. Further research into the mechanism of target cell recognition by bacterial bacteriolytic proteases will enable the use of this knowledge to expand the specificity of action of these enzymes, including as antimicrobial agents for medical applications. Full article

Rapid alkalinization factor (RALF) peptides are recognized as multifunctional regulators of plant stress responses, yet their roles in woody species remain poorly defined. Here, we identified a RALF22-like peptide from poplar ‘Shanxin’ (Populus davidiana × P. bolleana; PdbRALF22-like) and investigated its roles in salt tolerance and disease resistance. Synthetic PdbRALF22-like peptide elicited a rapid ROS burst in poplar leaf discs. In Nicotiana benthamiana, which was otherwise unresponsive to the peptide, transient expression of either of two poplar FERONIA-like receptor kinases (PdbFER-like-1 and PdbFER-like-2) enabled peptide-triggered ROS production, consistent with receptor-matched responsiveness in a heterologous context. Using CRISPR/Cas9, we generated a PdbRALF22-like knockout line and assessed salt tolerance in vitro and soil-grown assays. Under salinity, the mutant showed sustained rooting at high NaCl concentrations and improved growth relative to wild type. After 0.2 M NaCl treatment, soil-grown mutant plants exhibited reduced wilting and leaf injury. Evans Blue, DAB, and NBT staining indicated reduced membrane damage and lower accumulation of hydrogen peroxide and superoxide in the mutant. Significantly, the same knockout line displayed increased susceptibility to infection by the poplar leaf spot fungus, with larger lesions and higher pathogen biomass, accompanied by reduced ROS output and lower induction of the defense marker gene PdbPR1. Collectively, PdbRALF22-like negatively regulates salt tolerance while contributing positively to disease resistance, and represents a regulatory node linking salinity tolerance and disease susceptibility in poplar ‘Shanxin’, with poplar FER-like receptors providing a plausible route for peptide-triggered ROS signaling. This work expands our understanding of RALF peptide signaling in woody plants. Full article

Background: Cardiolipin (CL) is a phospholipid composed of a glycerol linked with two phosphatidate moieties that constitutes an integral part of the human inner mitochondrial membrane under physiological conditions. It is also vital for bacterial membrane transport and key bacterial functions associated with cell division and infection. CL is released in the cytosol or into the extracellular milieu upon cell death and during inflammation. We therefore tested the ability of CL to activate and expand tumor infiltrating lymphocytes (TIL) from patients with epithelial cancer. Methods: TIL were isolated from gastrointestinal tumor tissues and expanded in vitro in the presence of CL. The role of the NLRP3 inflammasome was evaluated using the specific inhibitor MCC950 and siRNA-mediated silencing of NLRP3. Phenotypic changes and T-cell potency were assessed via CXCL9/10 expression levels. To characterize the immune repertoire, deep TCR sequencing was performed to compare the TCR Vα and Vβ CDR3 regions between TIL and the corresponding tumor tissue. Recognition of autologous tumor cells and tumor-specific mutations, including mutations in KRAS and mitochondrial UQCRFS1 (D145V), was assessed using MHC class I and II restriction assays. Results: CL-expanded TIL exhibited increased CXCL9/10 expression, which is associated with increased potency of tissue invasion. CL-TIL exhibited broader recognition of frequently occurring KRAS mutations, and this effect could be blocked with an inhibitor (MCC950) of the NLRP3 pathway, a multiprotein inflammatory complex associated with danger signaling. TIL exhibited an enriched TCR Vα and Vβ CDR3 repertoire compared to tumor tissue, as defined by deep TCR sequencing. TCR αβ+ TIL recognized autologous tumor tissue in an MHC class I– and class II–restricted fashion, including the mutant HLA-DP–restricted mitochondrial protein associated with the electron respiratory chain complex III (UQCRFS1 D145V) presented by autologous tumor cells. Conclusions: CL activates the NLRP3 inflammasome pathway in TIL from patients with GI cancer and increases CXCL9/CXCL10 expression in TIL, resulting in enhanced recognition of mutant cancer–associated target epitopes, including a mitochondrial protein. CL may provide a danger signal: that facilitates TIL expansion via CL-activated pathways. Full article

Antimicrobial peptides (AMPs) are natural bioactive peptides produced by all organisms—from plants to insects, microbes and animals—and constitute a first line of defense. As they exhibit a broad spectrum of activity (antibacterial, antiviral, antifungal, antiparasitic, anticancer), strong efforts are being made to integrate AMPs into clinical use. AMPs are also being investigated as anticancer agents to overcome the side effects and/or resistance associated with current chemotherapies. In this context, we identified the natural AMP chionodracine from a new biological source: an Antarctic fish. Starting from the fragmentation of a chionodracine mutant peptide, a rational modular design approach was applied to develop three very short peptides (Pep-A, Pep-B and Pep-C), which were further modified with an N-terminal myristic acid lipid tail. The anticancer activity of the three N-myristoylated short peptides (Myr-A, Myr-B and Myr-C) was explored against the human cervical cancer HeLa cell line. The rationale behind this study is based on the previously reported antifungal activity of these myr peptides and on their ability to interact selectively with biological membrane-mimicking synthetic phospholipids without being particularly hemolytic or cytotoxic towards normal cells. We first demonstrated that myr peptides had cytotoxic activity against HeLa cells (IC₅₀ from 32 to 47 μM) but spared healthy primary human fibroblasts, whereas the corresponding non-myr peptides failed to kill cancer cells. The peptide with no hemolytic activity and a low IC₅₀, labeled Myr-B, was selected for subsequent analyses. Lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM) analysis revealed membrane damage and predominantly necrotic cell death in HeLa cells exposed to IC₅₀ doses of the Myr-B peptide, compared with cells treated with Pep-B. To thoroughly investigate the molecular effects of Myr-B in HeLa cells, we employed high-resolution label-free shotgun quantitative proteomics coupled with bioinformatics. Our results showed that exposing HeLa cells to Myr-B led to the under-expression of proteins belonging to the “apoptosis- and splicing-associated protein complex”, potentially influencing the alternative splicing process and consequently leading to a possible susceptibility to programmed cell death. These findings indicate that modifying natural AMPs may be a promising strategy for developing selective anticancer drugs and pinpoint Myr-B as an interesting target for future studies. Full article

CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling. We previously found that UV cross-linking of proteins from HeLa cervical cancer cells resulted in STAT3 interacting with the CARP-1 (614–638) peptide. Mutagenesis and co-IP-WB experiments revealed that CARP-1 interacts with a 40-amino-acid epitope from positions 441–480 (CE Epitope) located in the STAT3 DNA-binding domain. Overexpression of mutant STAT3 with in-frame deletion of the CE epitope (Gst-STAT3 (ΔCE) mutant), but not Gst-STAT3 (WT), failed to translocate to the nucleus in IL-6-treated cells. The small GTPase p21Rac1 interacts with and regulates STAT3 activation and nuclear translocation. Here we report the interaction of p21Rac1 with the CE epitope of STAT3 and the CARP-1 (600–650) region, suggesting that CARP-1 is part of a dynamic STAT3-p21Rac1 complex that functions in STAT3 activation and nuclear translocation. Expression of a STAT3 (ΔCE) mutant abolished STAT3 Y705 phosphorylation in cells that were treated with EGF or IL-6. Fine mapping revealed that scrambling the CE epitope peptide or a small peptide from positions 456–465 within the CE epitope resulted in abrogation of STAT3 Y705 phosphorylation by IL-6. Moreover, STAT3 phosphorylation by EGF or IL-6 was diminished in multiple CARP-1 null cancer cells. Importantly, incubation of a TAT-tagged STAT3 (454–467) peptide but not its scrambled version resulted in a reduction in STAT3 Y705 phosphorylation by IL-6/EGF. Taken together, our data demonstrates that the STAT3 CE epitope interacts with CARP-1 and p21Rac1, harbors novel sequences that activate STAT3 and promotes its nuclear translocation by IL-6/EGF. Full article

Histidine triad (HIT) family proteins contain a conserved histidine triad motif and play key roles in fungal metabolism and pathogenicity. This study focused on VD9136, a member of the HIT family in Verticillium dahliae, aiming to elucidate its biological function and mechanism underlying its role in cotton pathogenesis. A systematic investigation of the VD9136 gene in V. dahliae was conducted using bioinformatics analysis, gene knockout, genetic complementation, and pathogenicity assays. The results showed that VD9136 protein consists of 136 amino acids and is a stable, neutral, and weakly hydrophilic protein that lacks transmembrane domains and signal peptides; it is localized to the extracellular space via a non-classical secretion pathway. Its secondary structure is predominantly composed of α-helices and random coils. Phylogenetic analysis revealed that VD9136 is closely related to VliHIT, a homologous protein from V. longisporum, the pathogen responsible for Verticillium wilt in rapeseed. The promoter region of VD9136 contains multiple cis-acting elements, including light-responsive, hormone-responsive, and stress-responsive elements, indicating that its transcription may be regulated by multiple signaling pathways. VD9136 was significantly upregulated during the early stage of cotton infection (6–24 h post-inoculation). Pathogenicity assays demonstrated that V. dahliae knockout mutants lacking VD9136 exhibited a significant reduction in virulence, as evidenced by a lower disease index, decreased fungal biomass within plant tissues, and attenuated vascular browning in cotton plants. The pathogenic phenotype was successfully restored in genetic complementation strains. This study identified VD9136 as a key regulatory factor in the pathogenic process of V. dahliae, and its loss of function reduces the pathogenicity of V. dahliae. The findings provide a theoretical basis for elucidating the pathogenic mechanism of cotton Verticillium wilt and for developing corresponding prevention and control strategies. Full article

Sexual reproduction is the predominant mode of reproduction in animals, and spermatogenesis is the fundamental step in this process. As the model organism for lepidopteran, the silkworm Bombyx mori exhibits typical dichotomous spermatogenesis, producing both nucleated (eupyrene) and anucleate (apyrene) sperm. Leucylaminopeptidases (LAPs), members of the M17 metalloprotease family, are characterized by their ability to cleave leucine residues from the N-terminus of peptides. In addition to this canonical function, they have been implicated in male fertility in mammals and Diptera. Nevertheless, whether LAPs are required for dimorphic spermatogenesis in Lepidoptera remains to be clarified. Here, we demonstrated that Sperm-Leucylaminopeptidase (S-LAP) plays vital roles in the silkworm eupyrene sperm development. Similar to the testis-specific expression pattern of eight S-LAPs in Drosophila melanogaster, BmS-LAP was also predominantly expressed in testis. Depletion of BmS-LAP via CRISPR/Cas9 system resulted in male sterility, while the fertility of female mutant was unaffected. Notably, male mutants displayed severe defects in the formation and migration of eupyrene sperm, whereas apyrene sperm development appeared normal. In addition, RNA-seq and qRT-PCR analyses demonstrated that spermatogenesis defects were associated with energy metabolism and flagellar assembly. Our study provides the first evidence that LAP is necessary for dimorphic spermatogenesis in Lepidopteran, offering new insights into the molecular basis of male infertility. Full article

Melanoma is characterized by a high mutational burden making it an established model for studying tumor neoantigens and developing strategies for personalized immunotherapy. In this study, a reproducible bioinformatics pipeline was developed and implemented for the identification and prioritization of candidate neoantigens derived from non-synonymous somatic mutations in melanoma, using genomic data from the MSK-IMPACT cohort (mel-mskimpact-2020; n = 696) and comparative reference information from TCGA-SKCM. From the somatic mutation annotation file (MAF), 16,311 non-synonymous mutations were filtered, from which 50,480 mutant 8–11-mer peptides were generated using a sliding-window approach centered on the mutated position. Peptide–HLA class I binding affinity was predicted using MHCflurry 2.0 across six representative alleles (HLA-A*02:01, HLA-A*24:02, HLA-B*35:01, HLA-B*39:05, HLA-C*04:01, and HLA-C*07:02). Candidate prioritization was initially based on predicted binding percentile (rank ≤ 2), identifying 12,209 peptide–HLA combinations with high predicted binding affinity. To refine candidate selection, additional computational analyses were incorporated, including proteasomal cleavage prediction using NetChop 3.1 and estimation of T-cell epitope immunogenicity using the Immune Epitope Database (IEDB) immunogenicity predictor. Furthermore, a direct comparison between mutant (MUT) and corresponding wild-type (WT) peptides was performed using Δaffinity and Δrank metrics to evaluate the predicted impact of somatic mutations on HLA binding. The analysis revealed a predominance of peptides associated with the HLA-B locus, particularly the allele HLA-B*35:01, among the interactions with the lowest predicted binding percentiles. Several high-ranking peptide candidates were derived from genes with known roles in melanoma biology, including PLCG2, GATA3, AKT1, PTEN, PTCH1, and SMO. Overall, the integrative computational framework implemented in this study enables the systematic prioritization of candidate neoantigens derived from non-synonymous mutations in melanoma. This pipeline provides a reproducible strategy for exploring tumor neoantigen repertoires and may serve as a foundation for subsequent experimental validation and for studies related to neoantigen-based immunotherapies and immunopeptidomics. Full article

Streptococcus pneumoniae resists host defenses through multiple surface factors, yet their specific contribution to protection against antimicrobial peptides remains incompletely understood. We examined the role of pneumococcal surface protein A (PspA) and the polysaccharide capsule in protection against the human defensin HNP-1. PspA conferred increased resistance to HNP-1-induced killing, shown by a decreased killing in the presence of purified recombinant PspA and an increased sensitivity when PspA was deficient from the surface of strains of two different genetic backgrounds or when anti-PspA antibody was present. The capsule also conferred protection against HNP-1, which was serotype-dependent, with type 2 protecting better than type 4, and free polysaccharides acted as decoys by sequestering HNP-1. Removal of surface PspA from capsule-deficient mutants revealed additive contributions of both factors to survival. Molecular docking analysis suggests a potential electrostatic interaction between PspA and HNP-1. These findings highlight the independent and complementary roles of PspA and the capsule in pneumococcal resistance to HNP-1 and provide novel insights that may inform future vaccine design and antimicrobial strategies. Full article

Sterol regulatory element-binding proteins (SREBPs) regulate lipid homeostasis and coordinate sterol metabolism and carotenogenesis in the astaxanthin-producing yeast Phaffia rhodozyma. While Sre1, the SREBP ortholog, and the site-2 protease Stp1 have been identified as essential components of this pathway in P. rhodozyma, additional factors involved in Sre1 processing or regulation remain unknown. In Aspergillus species, a signal peptide peptidase contributes to the activation of the SREBP ortholog, raising the possibility of a similar role in this yeast. In this work, we identified and characterized the P. rhodozyma signal peptide peptidase (SppA) homolog. Sequence analysis, domain prediction, and phylogenetic analyses supported its classification within the SPP family of intramembrane aspartyl proteases. To evaluate its functional role, ΔsppA mutants were constructed in genetic backgrounds with constitutive Sre1 activity, including the cyp61⁻ mutant and a strain expressing the active form of Sre1 (Sre1N). Deletion of SPPA did not alter sensitivity to clotrimazole or cobalt chloride, nor affect pigmentation, indicating that SppA is not required for Sre1 activation in P. rhodozyma. Transcriptomic analyses further showed that expression of SRE1 and of its known target genes remained unchanged upon SPPA deletion. Interestingly, the loss of SppA in the Sre1N background caused marked downregulation of genes associated with protein refolding and unfolded protein binding. In agreement with these transcriptional changes, the Sre1NΔsppA strain displayed increased sensitivity to dithiothreitol. These findings suggest that, although SppA is not involved in Sre1 activation in P. rhodozyma, it may play a role in protein stress-related processes. Future studies will be required to define the molecular mechanisms underlying this role and its integration with protein homeostasis networks. Full article

Transport of Golgi-localized proteins from the ER is mediated by the coat protein complex II (COPII) and its members, COPII inner coat subunit Sec24 and Secretion-associated Ras-related GTPase 1 (Sar1). Sar1 and Sec24 recognize cytosolic N-termini of glycosyltransferases (GTs) that contain peptide signals required for incorporation into COPII-coated vesicles. Xyloglucan Xylosyltransferases (XXTs) are required for xyloglucan (XyGs) biosynthesis and must be transported to the Golgi for proper function. In this study, we demonstrated that XXTs interact with AtSar1 in the COPII complex but not with AtSec24, which was previously reported to be the main recruiter of cargo proteins into COPII-coated vesicles. The mutation of the arginine to glutamine residues of di-arginine motifs in the N-termini of XXTs caused protein mislocalization and significantly reduced the strength of the interaction with AtSar1. These mutations caused 90% of XXTs to either remain in the ER or localize to small non-Golgi compartments. In turn, such mislocalization significantly suppressed the recovery of XyGs biosynthesis in Arabidopsis thaliana (Arabidopsis) mutants (xxt1xxt2 and xxt3xxt4xxt5), failing to restore their root phenotypes to normal. Our results demonstrate the interaction between cargo and AtSar1, highlighting the critical role of di-arginine motifs in this interaction. These results provide new insights into the mechanism of ER-to-Golgi delivery of plant GTs, which significantly advances our understanding of polysaccharide biosynthesis in the Golgi and the enzymes responsible for it. Full article

Neoantigen vaccines have revitalized cancer vaccination by targeting tumor-specific mutant epitopes largely absent from central tolerance. Yet, clinical benefits remain inconsistent, in part because conventional vaccine platforms often do not reliably deliver antigens within an inflammatory tumor context, struggle to overcome immunosuppressive tumor microenvironments, and may not rapidly adapt to tumor heterogeneity and evolution. Oncolytic viruses (OVs) provide a mechanistically distinct route to “vaccinate in situ” by coupling tumor-selective infection and immunogenic cancer cell death with local innate immune activation, antigen release, and remodeling of the tumor microenvironment. In parallel, advances in sequencing, neoantigen prediction (e.g., updated NetMHCpan and MHCflurry tools as of 2025), and antigen presentation validation have enabled rational selection of patient-specific targets. At the same time, modern OV engineering supports insertion of neoantigen payloads and immune-modulatory transgenes. Here, we summarized principles that underpin neoantigen-encoded OVs as personalized cancer vaccines, emphasizing how OV adjuvanticity and antigenicity interact to drive priming, epitope spreading, and durable systemic immunity. We discussed major OV platforms with respect to payload capacity, expression control, manufacturability, and clinical track records, including lessons learned from approved or late-stage OVs such as talimogene laherparepvec (T-VEC) and teserpaturev/G47Δ. We also discussed design choices for encoding neoantigens (polyepitope strings, minigenes, long peptides; class I/II balancing), prioritizing translational biomarkers and immune-monitoring strategies, and outlining regulatory and GMP considerations for “platform-plus-variable insert” products. Finally, we propose a pragmatic clinical workflow for rapid personalization to maximize therapeutic index. Tightly integrating neoantigen science with immunovirotherapy, including recent 2025 preclinical advances like oncolytic adenovirus neoantigen delivery sensitizing low-TMB tumors to PD-1 blockade, could enable next-generation personalized cancer vaccines capable of converting “cold” tumors into responsive, systemically controlled disease. Full article

Aspergillus westerdijkiae is a common pathogenic fungus responsible for postharvest fruit rot in pears, causing substantial economic losses. This fungus also produces ochratoxin A (OTA), which poses serious health risks to humans. During host colonization, fungal pathogens secrete effectors to facilitate invasion. Under host-mimicking culture conditions, transcriptomic analysis of A. westerdijkiae at 24 and 72 h post-inoculation (hpi), combined with signal peptide prediction, identified 272 and 214 up-regulated secreted protein-encoding genes, respectively. Among these, a carboxylesterase gene, AwCES, was found to be significantly up-regulated. Compared to the wild-type strain, deletion of AwCES resulted in reduced conidial production and germination rate. Further studies revealed that the deletion mutant showed significantly attenuated virulence on pear fruit. Moreover, the loss of AwCES impaired fungal adaptation to stress environments. Collectively, these findings demonstrate that AwCES plays a critical role in the growth, development, and pathogenicity of A. westerdijkiae. Full article