The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.

Targeted therapeutics have transformed cancer treatment by selectively eliminating malignant cells while limiting systemic toxicity. L-asparaginase (L-ASNase), which induces metabolic stress by depleting asparagine (Asn), is clinically used for hematological malignancies but shows limited activity against solid tumors due to poor delivery and an immunosuppressive microenvironment. We previously developed CRT3LP, a calreticulin (CRT)-targeting monobody-L-ASNase fusion protein, designed to exploit immunogenic cell death (ICD); however, its therapeutic potential is constrained by insufficient immune activation. Here, we show that CRT3LFP, a multifunctional fusion protein incorporating the flagellin B subunit (FlaB) into the CRT3LP scaffold, effectively promotes M2-to-M1 macrophage polarization while maintaining tumor-selective metabolic disruption. In combination with the tumor-colonizing bacterial strain CNC018, which induces surface-exposed CRT, CRT3LFP achieves precise tumor localization. This synergistic approach significantly inhibits tumor growth and reshapes the tumor microenvironment, characterized by enhanced macrophage polarization, dendritic cell maturation, and CD8 ⁺ T-cell expansion. Additionally, CD47 blockade further potentiates this effect, fostering the formation of durable immune memory. Together, our findings establish CRT3LFP as a tumor-targeted immunometabolic platform that integrates metabolic deprivation with coordinated innate and adaptive immune activation to overcome resistance in solid tumors.

Targeted therapeutics have transformed cancer treatment by selectively eliminating malignant cells while limiting systemic toxicity. L-asparaginase (L-ASNase), which induces metabolic stress by depleting asparagine (Asn), is clinically used for hematological malignancies but shows limited activity against solid tumors due to poor delivery and an immunosuppressive microenvironment. We previously developed CRT3LP, a calreticulin (CRT)-targeting monobody-L-ASNase conjugate, designed to exploit immunogenic cell death (ICD); however, its therapeutic potential is constrained by insufficient immune activation. Here, we show that CRT3LFP, a multifunctional fusion protein incorporating the flagellin B subunit (FlaB) into the CRT3LP scaffold, potentially promotes M2-to-M1 macrophage polarization while maintaining tumor-selective metabolic disruption. In combination with the tumor-colonizing bacterial strain CNC018, which induces surface-exposed CRT, CRT3LFP achieves precise tumor localization and promotes M2-to-M1 macrophage polarization. This synergistic approach significantly inhibits tumor growth and reshapes the tumor microenvironment (TME), characterized by enhanced maturation of dendritic cells (DCs) and expanded CD8 ⁺ T cells. Furthermore, CD47-SIRPα blockade further potentiates this effect, leading to complete tumor eradication and the establishment of durable immune memory. Together, our findings establish CRT3LFP as a tumor-targeted immunometabolic platform that integrates metabolic deprivation with coordinated innate and adaptive immune activation to overcome resistance in solid tumors.

Cellular senescence is accompanied by widespread chromatin and transcriptional remodeling, but datasets that capture these changes across intermediate stages of replicative senescence remain limited. Here, we present an integrated bulk RNA-seq and ATAC-seq resource generated from primary human colonic fibroblasts spanning three replicative states: early-passage young cells, intermediate pre-senescent cells, and late-passage senescent cells. This serial design enables not only endpoint comparison between young and senescent cells but also stepwise evaluation of molecular changes during senescence progression. Bulk RNA-seq profiles captured transcriptome-wide alterations across the three states, while ATAC-seq defined corresponding changes in chromatin accessibility. By integrating promoter-associated accessibility with gene expression, we generated a gene-level multi-omic resource for evaluating concordant and discordant chromatin-transcription relationships across senescence transitions. The dataset also supports downstream analyses including pathway enrichment and transcription factor activity inference. Together, this dataset provides a useful resource for studying chromatin accessibility, transcriptional regulation, and their coupling during the progression of replicative senescence in human colonic fibroblasts.