Protein Family Research Articles

Chemoproteomics Sheds Light on Epigenetic Targets of [11C]Martinostat in the Human Brain.

Initiation of research programs to investigate binding specificity based on in vivo positron emission tomography (PET) imaging results can provide rich opportunities to improve data interpretation, gain biological insight, and inform hypothesis development. Here, we profile the binding specificity of the neuroepigenetic imaging probe, [11C]Martinostat. In vivo neuroimaging studies using [11C]Martinostat have uncovered differential regional uptake in relation to age and biological sex and in patients with schizophrenia, bipolar disorder, Alzheimer's disease, and low-back pain compared to healthy controls. Previous studies using recombinant proteins and thermal shift assays in postmortem tissue indicate that [11C]Martinostat engages class I and putatively class IIb histone deacetylases (HDACs). While HDACs serve multiple functions, including regulation of chromatin remodeling and gene transcription, it is not known how differences in HDAC expression may arise across brain regions. HDACs functionally interact with a diverse array of multisubunit complexes, and engagement with associated binding partners may contribute to these differences. To further assess target engagement of [11C]Martinostat, we designed a synthetic probe based on the inhibitor structural scaffold for use in competition experiments followed by proteomic analysis in postmortem tissue. The synthetic probe, called Compound 4, appears to interact with the class I HDAC paralog HDAC2 and the class IIb paralog HDAC6 in a robust manner. We also uncovered unique interacting partners, including synaptic proteins from the synaptotagmin (SYT) family of proteins and neuronal pentraxin 2 (NPTX2). Further work to investigate HDAC associations with interacting proteins across regions of the human brain is needed to better understand neuroepigenetic dysregulation in psychiatric and neurological conditions.

SORFdb – a database for sORFs, small proteins, and small protein families in bacteria

Small proteins with fewer than 100, particularly fewer than 50, amino acids are still largely unexplored. Nonetheless, they represent an essential part of bacteria’s often neglected genetic repertoire. In recent years, the development of ribosome profiling protocols has led to the detection of an increasing number of previously unknown small proteins. Despite this, they are overlooked in many cases by automated genome annotation pipelines, and often, no functional descriptions can be assigned due to a lack of known homologs. To understand and overcome these limitations, the current abundance of small proteins in existing databases was evaluated, and a new dedicated database for small proteins and their potential functions, called ’sORFdb’, was created. To this end, small proteins were extracted from annotated bacterial genomes in the GenBank database. Subsequently, they were quality-filtered, compared, and complemented with proteins from Swiss-Prot, UniProt, and SmProt to ensure reliable identification and characterization of small proteins. Families of similar small proteins were created using bidirectional best BLAST hits followed by Markov clustering. Analysis of small proteins in public databases revealed that their number is still limited due to historical and technical constraints. Additionally, functional descriptions were often missing despite the presence of potential homologs. As expected, a taxonomic bias was evident in over-represented clinically relevant bacteria. This new and comprehensive database is accessible via a feature-rich website providing specialized search features for sORFs and small proteins of high quality. Additionally, small protein families with Hidden Markov Models and information on taxonomic distribution and other physicochemical properties are available. In conclusion, the novel small protein database sORFdb is a specialized, taxonomy-independent database that improves the findability and classification of sORFs, small proteins, and their functions in bacteria, thereby supporting their future detection and consistent annotation. All sORFdb data is freely accessible via https://sorfdb.computational.bio.

Epsin3 promotes non-small cell lung cancer progression via modulating EGFR stability

BackgroundThe abnormal expression and overactivation of the epidermal growth factor receptor (EGFR), a typical cancer marker for non-small cell lung cancer (NSCLC), are closely related to the tumorigenesis and progression of NSCLC. However, the endocytosis mechanism of EGFR in lung cancer is not yet known. Epsin3 (EPN3), a member of the endocytic adaptor protein family, is essential for the endocytosis of multiple receptors. In this study, we aimed to investigate the role of EPN3 in modulating EGFR function, its effects on NSCLC progression, and its potential involvement in tyrosine kinase inhibitor (TKI) resistance, which remains a significant hurdle in NSCLC treatment.ResultsOur findings revealed that the expression of EPN3 is significantly up-regulated in NSCLC patients. Elevated EPN3 expression was proportional to shorter overall survival in patients with NSCLC. Functional analyses revealed that EPN3 directly interacts with EGFR, enhancing its recycling to the plasma membrane and preventing its degradation via the lysosomal pathway. This stabilization of EGFR led to sustained downstream signalling, promoting NSCLC cell proliferation and migration. Notably, mutations in the EGFR tyrosine kinase domain, which typically confer resistance to TKIs, did not alter the regulatory effect of EPN3.ConclusionsEPN3 enhances EGFR signalling by promoting its recycling and stability, contributing to NSCLC progression and TKI resistance. Targeting EPN3 could offer a novel therapeutic strategy to overcome drug resistance in EGFR-driven NSCLC.

Selection Pressure Regulates the Evolution-Structure-Function Paradigm of Monocyte Chemoattractant Protein Family.

Monocyte chemoattractant proteins (MCPs) are involved in monocyte trafficking during severe inflammation by modulating the chemokine-glycosaminoglycan-receptor signaling axis. MCPs comprise a family of four chemokines (CCL2, CCL7, CCL8, and CCL13/12) that exhibit differential expression patterns in mammals, functional diversity, and receptor/glycosaminoglycan (GAG) binding promiscuity. In this context, the evolution-structure-function paradigm of MCP chemokines in mammals was established by assessing phylogeny, functional divergence, selection pressure, and coevolution in correlation with structural and surface characteristics. Comprehensive analyses were performed using an array of evolutionary and structural bioinformatic methods including molecular dynamics simulations. Our findings demonstrate that substitutions in receptor/GAG-interacting residues mediate episodic diversification and functional diversity in MCP chemokines. Additionally, a balanced interplay of selection pressures has driven the functional changes observed among MCP paralogs, with positive selection at various receptor/GAG-binding sites contributing to their promiscuous receptor/GAG interactions. Meanwhile, processes like purifying selection and coevolution maintain the classical chemokine structure and preserve the ancestral functions of MCP chemokines. Overall, this study suggests that selection pressure on sites within the N-terminal region [N-loop and 310-helix] and 40S loop of MCP chemokines alters surface properties to fine-tune the molecular interactions and functional characteristics without altering the overall chemokine structure.

WD repeat domain 4 in tumorigenesis: Molecular mechanisms, cancer-type specific roles, and therapeutic potential

WD repeat domain 4 (WDR4) is an essential member of the WD-repeat protein family, known for its regulatory roles in cellular processes critical to cancer development, including RNA modification, protein stability, cell cycle progression, and apoptosis. Studies have shown that WDR4 plays a pivotal role in tumorigenesis across various cancer types, with a particular focus on breast cancer in this study, where its overexpression is closely associated with aggressive tumor characteristics and poorer patient outcomes. As a scaffold protein, WDR4 is involved in N7-methylguanosine tRNA methylation and ubiquitin-mediated protein degradation, thereby regulating RNA stability, protein synthesis, and cell survival. This review provides a comprehensive analysis of WDR4’s molecular mechanisms, its oncogenic functions across different cancer types, and its interactions with other key factors in the tumor microenvironment, further exploring its potential role in tumor progression. As research on WDR4 progresses, we not only gain a deeper understanding of its complex role in tumor biology but also uncover new therapeutic avenues. In particular, the potential of WDR4 as a biomarker and therapeutic target is increasingly recognized. Despite the challenges faced in its clinical application, such as the difficulty in developing targeted therapies and managing side effects, the future prospects of WDR4 in cancer diagnosis and treatment remain promising, and it is expected to emerge as an effective therapeutic target in the near future.

Exploring BCL2 regulation and upstream signaling transduction in venetoclax resistance in multiple myeloma: potential avenues for therapeutic intervention

Investigating venetoclax (VTX) resistance in multiple myeloma (MM) is crucial for the development of novel therapeutic strategies to tackle resistance. We conducted a multi-omic characterization of established VTX-resistant isogenic human myeloma cell lines (HMCL) and primary MM patient samples pre- and post-VTX treatment. Transcriptomic and proteomic analysis revealed that resistance was largely associated with BCL-2 family protein dysregulation, including upregulation of anti-apoptotic proteins such as MCL-1, BCL-XL, BCL-2, and downregulation of pro-apoptotic members. Notably, the re-introduction of BIM into resistant cells restored VTX sensitivity and synergized with MCL-1 inhibitors. Upstream signaling pathways, including growth factor receptor tyrosine kinase (RTK) and phosphoinositide-3-kinase (PI3K) were implicated in this dysregulation. Simultaneous inhibition of MCL-1, BCL-XL, and upstream PI3K, RTK (FGF, EGF, and IGF1) mediated signaling enhanced VTX sensitivity. Post-translational modifications of MCL-1, particularly its stabilization via acetylation and phosphorylation, were investigated, although their inhibition only marginally increased VTX sensitivity. Lastly, the inhibition of AURKA and mitochondrial respiration also improved VTX sensitivity in some resistant HMCLs. Our findings suggest that combining VTX with MCL-1 and BCL-XL inhibitors or PIK3/RTK inhibitors holds potential for overcoming resistance. The study illustrates the importance of understanding molecular determinants of resistance to develop tailored therapeutic strategies.

BRD4 Induces Esophageal Squamous Cell Carcinoma Progression via the Wnt/β-catenin Pathway.

BRD4, part of the bromodomain and extra terminal domain (BET) protein family, plays a pivotal role in gene transcription, DNA replication, and repair via transcription regulators. Despite its established involvement in various human diseases, its function in esophageal squamous cell carcinoma (ESCC) has not been fully explored. Our research investigated the association of BRD4 in ESCC and its underlying molecular mechanisms. The findings revealed that BRD4 knockdown notably diminished the cells' proliferation, migration, invasion capabilities and induced apoptosis and cell cycle arrest. Conversely, overexpression of BRD4 can reverse these phenotypes. Pearson correlation and enrichment analyses indicated that BRD4 expression was associated with the cell cycle and Wnt/β-catenin signaling pathway. Further validation confirmed that reduced BRD4 expression downregulates Cyclin D1 and c-Myc, and suppresses epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin signaling pathway. Furthermore, rescue experiments showed that overexpressing c-Myc significantly mitigated the inhibitory impact of BRD4. Moreover, by employing single-cell transcriptome sequencing, we explored the impact of the tumor microenvironment on BRD4 overexpression in ESCC cells. These insights confirmed BRD4's potential as a therapeutic target, suggesting that modulating its expression could yield promising strategies for ESCC treatment.

The DNA binding of plant-specific GROWTH-REGULATING FACTOR transcription factors is stabilized by GRF-INTERACTING FACTOR coactivators.

The plant-specific GROWTH-REGULATING FACTOR (GRF) transcription factor family proteins play crucial role in regulating diverse aspects of plant life. The transcriptional activity of GRFs is known to be enhanced through direct interaction with the GRF-INTERACTING FACTOR (GIF) coactivators. However, it remains unclear how the binding to GIF affects the biochemical ability of GRFs. Herein, we present evidence that GIFs also stabilize the DNA binding of GRFs. A combination of biochemical experiments and AlphaFold-predicted structural models suggests that the GIF-binding domain in GRFs may partially restrict their own DNA binding through direct interaction with the DNA-binding domain. These findings deepen our understanding of the GRF:GIF module in plant regulation and provide a basis for strategies to manipulate this module for agricultural and biotechnological applications.

Dnajc5b contributes to male fertility by maintaining the mitochondrial functions and autophagic homeostasis during spermiogenesis

DnaJ heat shock protein family member C5 beta (DNAJC5B), also known as cysteine-string protein beta, exhibits a prominent expression in testicular tissue and plays an important role in acrosomal exocytosis in vitro. Nevertheless, the precise role and underlying mechanism of DNAJC5B in spermatogenesis and male fertility remain poorly understood. The meta-analysis of RNA-sequencing datasets from porcine and murine testes reveals that Dnajc5b could be a pivotal factor in spermatogenesis. This study illustrates that male fertility declines with an increased ratio of abnormal spermatozoa in germ-cell knockout Dnajc5b mice. DNAJC5B has been identified as a mitochondrial protein with high expression in spermatids. The absence of DNAJC5B induces a cascade of mitochondrial damages, including oxidative stress, mitochondrial stress in the testes, and lower mitochondrial membrane potential of spermatozoa. In vivo and in vitro evidence demonstrates that DNAJC5B mitigates excessive cellular autophagy and mitophagy via DNAJ domain under environmental stress conditions, such as starvation or exposure to mitochondrial uncouplers FCCP and CCCP. This study highlights the important role of DNAJC5B in safeguarding male fertility by preserving mitochondrial function and regulating autophagy during spermiogenesis.

Tight junction proteins in glial tumors development and progression

Tight junctions form a paracellular barrier in epithelial and endothelial cells, and they regulate the diffusion of fluids, molecules, and the penetration of cells across tissue compartments. Tight junctions are composed of a group of integral membrane proteins, which include the claudin family, tight junction-associated Marvel protein family, junctional adhesion molecule family, and proteins that anchor the cytoskeleton, such as zonula occludens proteins and the cingulin family. Several factors, such as neurotransmitters or cytokines, and processes like ischemia/hypoxia, inflammation, tumorigenesis, phosphorylation/dephosphorylation, ubiquitination, and palmitoylation, regulate tight junction proteins. Claudins are involved in tumorigenesis processes that lead to glioma formation. In gliomas, there is a noticeable dysregulation of claudins, occludin, and zonula occludens-1 abundance, and their dislocation has been observed. The weakening of intercellular adhesion and cell detachment is responsible for glioma infiltration into surrounding tissues. Furthermore, the paracellular permeability of the blood–brain barrier, formed with the involvement of tight junction proteins, influences the development of peritumoral edema – and, simultaneously, the rate of drug delivery to the glial tumor. Understanding the junctional and paracellular environments in brain tumors is crucial to predicting glial tumor progression and the feasibility of chemotherapeutic drug delivery. This knowledge may also illuminate differences between high and low-grade gliomas.

A STING-CASM-GABARAP pathway activates LRRK2 at lysosomes.

Mutations that increase LRRK2 kinase activity have been linked to Parkinson's disease and Crohn's disease. LRRK2 is also activated by lysosome damage. However, the endogenous cellular mechanisms that control LRRK2 kinase activity are not well understood. In this study, we identify signaling through stimulator of interferon genes (STING) as an activator of LRRK2 via the conjugation of ATG8 to single membranes (CASM) pathway. We furthermore establish that multiple chemical stimuli that perturb lysosomal homeostasis also converge on CASM to activate LRRK2. Although CASM results in the lipidation of multiple ATG8 protein family members, we establish that LRRK2 lysosome recruitment and kinase activation are highly dependent on interactions with the GABARAP member of this family. Collectively, these results define a pathway that integrates multiple stimuli at lysosomes to control the kinase activity of LRRK2. Aberrant activation of LRRK2 via this pathway may be of relevance in both Parkinson's and Crohn's diseases.

Distinct Impact of Processing on Cross-Order Cry1I Insecticidal Activity

The insecticidal Cry proteins from Bacillus thuringiensis are used in biopesticides or transgenic crops for pest control. The Cry1I protein family has unique characteristics of being produced during the vegetative rather than sporulation phase, its protoxins forming dimers in solution, and exhibiting dual toxicity against lepidopteran and coleopteran pests. The Cry1Ia protoxin undergoes sequential proteolysis from the N- and C-terminal ends, producing intermediate forms with insecticidal activity, while in some cases, the fully processed toxin is inactive. We investigated the oligomerization and toxicity of Cry1Ia intermediate forms generated through trypsinization (T-Int) and larval gut fluid (GF-Int) treatments, as well as the fully trypsinized protein (toxin). Heterologously expressed intermediate forms assembled into oligomers and showed similar toxicity to Cry1Ia protoxin against Ostrinia nubilalis (European corn borer) larvae, while the toxin form was ~30 times less toxic. In contrast, bioassays with Leptinotarsa decemlineata (Colorado potato beetle) larvae did not show significant differences in toxicity among Cry1Ia protoxin, T-Int, GF-Int, and fully processed toxin. These results suggest that the Cry1I mode of action differs by insect order, with N-terminal cleavage affecting toxicity against lepidopteran but not coleopteran larvae. This knowledge is essential for designing pest control strategies using Cry1I insecticidal proteins.

Deciphering molecular specificity in MCL-1/BAK interaction and its implications for designing potent MCL-1 inhibitors.

The intricate interplay among BCL-2 family proteins governs mitochondrial apoptosis, with the anti-apoptotic protein MCL-1 primarily exerting its function by sequestering the pore-forming effector BAK. Understanding the MCL-1/BAK complex is pivotal for the sensitivity of cancer cells to BH3 mimetics, yet the precise molecular mechanism underlying their interaction remains elusive. Herein, we demonstrate that a canonical BH3 peptide from BAK inadequately binds to MCL-1 proteins, whereas an extended BAK-BH3 peptide with five C-terminal residues exhibits a remarkable 65-fold increase in affinity. By elucidating the complex structures of MCL-1 bound to these two BAK-BH3 peptides at 2.08 Å and 1.98 Å resolutions, we uncover their distinct binding specificities. Notably, MCL-1 engages in critical hydrophobic interactions with the extended BAK-BH3 peptide, particularly at an additional p5 sub-pocket, featuring a π-π stacking interaction between MCL-1 Phe319 and BAK Tyr89. Mutations within this p5 sub-pocket substantially disrupt the MCL-1/BAK protein-protein interaction. Furthermore, the p5 sub-pocket of MCL-1 significantly influences the efficacy of MCL-1 inhibitors. Overall, our findings elucidate the molecular specificity underlying MCL-1 binding to BAK and underscore the significance of the p5 hydrophobic sub-pocket in their high-affinity interaction, thus providing novel insights for the development of BH3 mimetics targeting the MCL-1/BAK interaction as potential therapeutics for cancer treatment.

Proteomic analysis and effects on osteogenic differentiation of exosomes from patients with ossification of the spinal ligament

Abstract Ossification of the spinal ligament (OSL), including ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF), is a multifactorial disease that includes genetic predisposition. The association between the rate of ossification in the spinal canal and the severity of myelopathy symptoms is well known, but the degree of progression varies widely among patients. Although many candidate genes and biomarkers have been reported, there are no definitive and quantitative conclusions to date, probably because of low reproducibility due to individual differences. In this study, we focused on exosomes secreted by ossified spinal ligament cells. Exosomes are crucial for intercellular communication during development and progression of disease. In a co-culture study of non-OLF cells with OLF cells, there was increased osteogenic differentiation, including Runx2 and Wnt3a expression, with use of exosome-penetrating filters (1.2 μm) compared to exosome-non-penetrating filters (0.03 μm). Dose-dependent increases in alkaline phosphatase activity and mineral deposition were observed in non-OLF cells treated with OLF-derived exosomes. These results support the hypothesis that OLF-derived exosomes are involved in regulation of osteogenic differentiation. In comparative proteomics analysis, 32 factors were increased and 40 were decreased in OLF-derived exosomes compared to non-OLF-derived exosomes. Molecular network analysis of these 72 factors indicated 10 significant pathways, including the MMP signaling, mTOR signaling, Wnt signaling and VDR-associated pathways. Among the upregulated exosomal membrane proteins in OLF samples, COL IV, FMNL3, mTORC2, and PIP4K showed increased expression with greater ossification, suggesting they may serve as biomarkers of disease activity and therapeutic targets. These factors are involved in the PI3K/Akt/mTOR signaling pathway, and particularly mTOR is known to regulate osteogenic and chondrogenic differentiation. In contrast, FABP5, several KRT family proteins, S100A8, SERPINB3, and TGM, were significantly downregulated in OLF-derived exosomes. These findings provide novel insights into the molecular mechanisms underlying OSL pathogenesis.

MicroRNA-Mediated Changes in DNA Methylation Affect the Expression of Genes Involved in the Thickness-of-Pod-Canopy Trait in Brassica napus L

Methylation plays an important role in regulating crop development, but little is known about how methylation regulates plant architecture in rapeseed (Brassica napus L.). Here, we examined how methylation affects the thickness-of-pod-canopy (TPC) trait in rapeseed by performing genome-wide methylation analysis of two extreme TPC lines. In flower buds, 26 genes had significantly higher methylation levels in the high-TPC samples compared to the low-TPC samples, resulting in significantly reduced gene expression. By contrast, in the stem apex samples, the promoter regions of 22 genes were hypermethylated in the high- vs. low-TPC samples. The promoters of 19 and 21 genes had significantly reduced methylation levels in the flower bud and stem apex, respectively, of the high- vs. low-TPC samples, resulting in significantly higher expression levels. Some of these differentially expressed genes are associated with TPC-related traits, such as BnaC01g12960D (NRT1.8). In addition, 14 important genes related to growth and development were differentially regulated between the two groups due to miRNA-mediated differences in methylation levels in their promoters. For example, hypermethylation in the promoter region of BnaCnng64040D (Lipase family protein), mediated by miR159, led to significantly reduced gene expression in flower buds of high-TPC vs. low-TPC lines. These results, together with our previously generated RNA-seq and miRNA profiling data, indicate that both methylation and miRNAs are perhaps involved in regulating the expression of genes, thereby affecting the TPC trait in B. napus, providing a reference for uncovering the molecular mechanism regulating this crucial trait.

Functional characterization of MMAR_1296 in Mycobacterium marinum and its potential as a vaccine candidate.

The Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in mycobacteria plays a crucial role in pathogenesis and immune evasion. These proteins characterized by unique structures with conserved sequences. This study elucidated the specific immunological functions of MMAR_1296 from marine mycobacterium. Expressing MMAR_1296 in Mycobacterium smegmatis (M. smegmatis) led to significant alterations in bacterial morphology, as well as reduced survival of M. smegmatis under adverse in vitro conditions and within macrophages. Furthermore, transcriptome analysis of mouse macrophages indicated that natural immunity-related pathways were upregulated in the group infected with M. smegmatis recombinantly expressing MMAR_1296. Moreover, the mycobacterium Growth Inhibition Assays(MGIA)in mice demonstrated that M. smegmatis expressing MMAR_1296 exerted a significant inhibitory effect against Mycobacterium abscessus (M. abscessus) and Mycobacterium marinum (M. marinum) infections. Immunization challenge experiments in mice further confirmed its protective effects, showing a reduction in organ bacterial loads by 1 log10 value compared to the positive control group. These findings indicate that MMAR_1296 is a promising vaccine candidate for M. abscessus and M. marinum. Given that PE/PPE protein family is also a crucial component of Mycobacterium tuberculosis (M. tuberculosis) antigens, further exploration of sequence functions based on MMAR_1296 could reveal broader applications of PE/PPE proteins family for M. tuberculosis treatment. This study supported vaccine development targeting PE/PPE proteins in mycobacteria and paves the way for broader applications.

Genome-wide identification of the WD40 protein family and functional characterization of AaTTG1 in Artemisia annua.

Sweet wormwood (Artemisia annua), an annual herb belonging to the Compositae family, is the main source of the potent anti-malarial drug artemisinin, which is mainly produced in glandular trichomes of A. annua leaves. The WD40 protein family is one of the largest protein families in eukaryotes and plays crucial roles in regulating plant growth and development, stress responses, and secondary metabolite biosynthesis. However, WD40 proteins have not been comprehensively identified in A. annua. In this study, we identified 236 WD40 proteins in the A. annua genome and examined their conserved domains, motifs, and cis-regulatory elements, gene structures, chromosomal distribution, duplication events of their encoding genes. Furthermore, we isolated and characterized TRANSPARENT TESTA GLABROUS 1 (AaTTG1), a homolog of Arabidopsis TTG1, and confirmed that AaTTG1 was localized to the nucleus and cytoplasm. Indeed, AaTTG1 can rescue the glabrous phenotype of the Arabidopsis ttg1 mutant and enhanced trichome production when heterologously expressed in wild-type Arabidopsis plants. Transgenic A. annua lines overexpressing AaTTG1 displayed a significantly higher density of glandular trichomes and higher artemisinin contents. Transgenic A. annua lines with inhibited AaTTG1 function had fewer glandular trichomes and lower artemisinin levels. Moreover, we demonstrated that AaTTG1 positively regulates glandular trichome development in A. annua through interactions with AaSPL9. This study thus provides fundamental insights into the role of WD40 proteins in A. annua and introduces a promising approach to enhance artemisinin production by manipulating glandular trichome development in this valuable medicinal plant.

The RNA-Binding Properties of Annexins.

Annexins are a family of calcium-dependent phospholipid-binding proteins involved in crucial cellular processes such as cell division, calcium signaling, vesicle trafficking, membrane repair, and apoptosis. In addition to these properties, Annexins have also been shown to bind RNA, although this function is not universally recognized. In the attempt to clarify this important issue, we employed an integrated combination of experimental and computational approaches. Using the catRAPID algorithm, we accurately predicted known RNA-binding partners of Annexins, supported by experimental validation. We then constructed a virtual library of potential mRNA partners for Annexin A2, identifying regions within its structure directly involved in RNA binding. Beyond RNA interaction, some Annexins, notably AnxA7 and AnxA11, exhibit strong phase separation tendencies driven by their N-termini. These biophysical properties likely play roles in RNA trafficking and localization particularly in neurons, where they may influence processes such as synaptic plasticity, learning, and memory. Our predictions contribute to a deeper understanding of the Annexin function, emphasizing their potential impact on RNA regulation and cellular compartmentalization through phase separation and propose a powerful computational tool for the prediction of RNA-binding properties.

Claudin 18.2 determination in locally advanced gastric and gastroesophageal adenocarcinoma treated with neoadjuvant chemotherapy.

492 Background: Gastric and esophago-gastric adenocarcinoma (GAC) represent a major unmet need. For tumors amenable with curative intent, prognosis remains poor with 5-year survival rates below 40%. Several strategies have been added to surgery including peri-operative chemotherapy which represents the standard of care for locally advanced GAC. Claudins is a family of transmembrane junctional proteins which acts in paracellular permeability, transport and communication. Claudin 18.2 (CLDN), a tissue-specific variant, is physiologically expressed in both gastric mucosa and gastric cancer. In metastatic GAC, CLDN positivity settles around 40% of cases and the addition of target therapy zolbetuximab to chemotherapy resulted in a significant improvement in overall survival. However, limited data are present about CLDN in early-stage disease. The aim of our study is to assess rates of expression of CDLN in locally advanced GAC treated with neoadjuvant chemotherapy and correlate it with clinical outcomes. Methods: We conducted a multi-omics prospective trial at IEO enrolling patients with locally advanced GAC treated with peri-operative chemotherapy and surgery. Pre-chemotherapy endoscopic samples were selected for CDLN determination. Positive cases were defined as moderate-to-strong membranous staining in ≥ 75% of tumor cells by IHC using the VENTANA CLDN18 (43-14A) Assay. CLDN expression was analyzed for correlation with pathologic complete response (pCR), tumor regression grade (TRG), event‐free survival (EFS), and overall survival (OS). Survival was calculated using Kaplan–Meier method and log-rank tests. Statistical analyses were performed using SPSS software (version 29.0.2.0). Results: We included 48 patients, 67% male, median age 62 years (35-78). 31 tumors originated from gastric body, 14 from the junctional region and 4 from distal esophagus. Most frequent histotypes were diffuse (43%) and tubular (39%). Five cases (11%) presented microsatellite instability. All patients received neoadjuvant chemotherapy, mainly FLOT (92%) and underwent surgery, with 93% of R0 resection. Three patients (6%) achieved pCR and 18 had a TRG 1A or B. Of the total, 20 cases were defined as positive (42%). After a median follow-up of 20 months, 12 patients relapsed and 3 died. No significant association was observed for CDLN positivity and pCR or TRG1 rate, as well as for primary site or histotype. PFS and OS were not statistically different according to claudin status, even though numerically shorter in CDLN positive cases (PFS: 31 vs 33 months, p=.43; OS: 36 vs 39 months, p =.10). Conclusions: In our study of locally advanced GAC, rate of CDLN positivity was 40%, similarly to advanced stage. However, no differences in pCR rate, regression grade and survival outcomes have been observed. Larger studies and longer follow-up are required to assess the impact of CDLN in early-stage GAC.

Berberine shaping the tumor immune landscape via pyroptosis.

Pyroptosis is a programmed cell death (PCD) mainly mediated by the Gasdermin family of proteins, among which Gasdermin E (GSDME) is considered a tumor suppressor gene. GSDME can recruit immune cells to the tumor microenvironment (TME) and promote their effects. Activating and enhancing adaptive immunity through GSDME is a potential solution for anti-tumor therapy. Here we reported that berberine (BBR), a small molecule from traditional Chinese medicine, as a GSDME activator, induced caspase-3 (C-3)/GSDME pathway-mediated pyroptosis through the mitochondrial pathway, improved the immunosuppressive state of the tumor microenvironment, and thus promoted anti-tumor immunity. We determined the induction of pyroptosis of 4T1 cells by BBR through various experiments, and investigated the immune activation effect of BBR by co-culture in vitro, which induced DCs maturation and macrophage polarization. Zebrafish embryo toxicity experiments were used to evaluate the in vivo safety of berberine. Furthermore, the in vivo antitumor and immune activation effects of BBR were investigated using 4T1 orthotopic model mice, and the results showed that BBR could eliminate orthotopic tumor cells by activating local and systemic immunity. Moreover, we observed that BBR significantly inhibited breast cancer lung metastasis. In summary, our results showd the role of BBR as a GSDME activator stimulated both local and systemic antitumor immune responses by inducing pyroptosis, effectively preventing tumor development and metastasis.