Biology subjects

Gude, S.

Publications and source records attributed to Gude, S..

3 recordsLinked to original sources

bioRxiv2025

Thioredoxin interacting protein (TXNIP), a redox regulator, mediates the RAPGEF3/4 signaling dependency in primary melanoma

Progression of cutaneous primary melanoma that arises from melanocytes leads to lethal metastatic disease. Molecular mechanisms that control the growth of primary melanoma in the skin and promote progression are not fully understood. Previously we showed that RAP guanine exchange factors EPAC1/2 (Exchange Protein Activated by cyclic AMP) promote the growth of primary melanoma and loss of dependency on EPACs is associated with metastatic progression. In this study, we show that EPACs are activated during malignant transformation of melanocytes, and chemical inhibition or genetic deletion of EPAC inhibits melanomagenesis in Braf/Pten mice. Low expression of EPAC mRNA and its effector RAP1-GTP protein in primary melanoma correlate with better recurrence-free survival. RNAseq analysis of matched primary and metastatic melanoma cells treated with an EPAC inhibitor showed that TXNIP, an important regulator of redox homeostasis, is a downstream effector of EPAC signaling. We also show that EPACs promote melanoma growth by regulating redox homeostasis and mitochondrial ROS through activation of mechanistic target of rapamycin complex 1 (mTORC1) that stabilizes hypoxia-inducible factor 1-alpha (HIF-1), a transcriptional activator of redox regulator TXNIP and glycolytic enzymes. Our data suggest that targeting mechanisms that melanoma cells employ to bypass EPAC dependency is a potential therapeutic approach. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/696903v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1967afaorg.highwire.dtl.DTLVardef@63b907org.highwire.dtl.DTLVardef@1089da4org.highwire.dtl.DTLVardef@a5b2ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Iterative in vivo cut'n'paste of chromosomal loci in Escherichia coli K-12 using synthetic DNA

We have developed an easy-to-use in vivo method to iteratively relocate functional chromosomal loci onto an episome in Escherichia coli by utilizing synthetic DNA fragments. In this in vivo cutnpaste procedure, "cutting" is executed by the RNA-guided DNA endonuclease Cas9 and a set of guides, while "pasting" is facilitated by the phage {lambda} Red recombinase, which are all synthesized on easily curable helper plasmids. To demonstrate the utility of in vivo cutnpaste, we commercially obtained synthetic DNA fragments containing locus-specific homology regions, antibiotic marker cassettes, and standardized Cas9 target sequences, and successfully relocated seven functional chromosomal loci. Scarless relocation mutants of the trg, aer, tsr, malHM, malQT, macB-nadA, and folA chromosomal loci were obtained as antibiotic-resistant isolates by combining Cas9 counterselection with the restoration of an antibiotic marker cassette. The additional antibiotic marker cassettes and standardized Cas9 target sequences present in the synthetic DNA fragments are inherently eliminated upon completion of the procedure, enabling iterative processing of chromosomal loci. In vivo cutnpaste should be widely useful, especially in genome (re-)engineering efforts of E. coli and other bacteria because the procedure can be performed by non-specialists in unmodified wild-type cells. SignificanceEditing of genomes is crucial to understand the inner workings of bacteria and to alter them for biotechnological applications. In vivo cutnpaste, in analogy to the frequently used keyboard shortcuts Ctrl-X and Ctrl-V which allow to move text, images, or files on computers, makes it easy to relocate large chunks of the bacterial genome without requiring skills to manipulate DNA outside of the bacterial cell. Like Ctrl-X and Ctrl-V, in vivo cutnpaste can be executed several times to iteratively edit multiple genomic locations in the same bacterium. Since in vivo cutnpaste entirely takes place inside the bacterial cell, it is particularly well suited to edit hard-to-tackle genomic features such as essential genes.

microbiology↗

bioRxiv2021

A salvaging strategy enables stable metabolite provisioning among free-living bacteria

All organisms rely on complex metabolites such as amino acids, nucleotides, and cofactors for essential metabolic processes. Some microbes synthesize these fundamental ingredients of life de novo, while others rely on uptake to fulfill their metabolic needs. Although certain metabolic processes are inherently leaky, the mechanisms enabling stable metabolite provisioning among microbes in the absence of a host remain largely unclear. In particular, how can metabolite provisioning among free-living bacteria be maintained under the evolutionary pressure to economize resources? Salvaging, the process of recycling and reusing, can be a metabolically efficient route to obtain access to required resources. Here, we show experimentally how precursor salvaging in engineered Escherichia coli populations can lead to stable, long-term metabolite provisioning. We find that salvaged cobamides (vitamin B12 and related enzyme cofactors) are readily made available to non-productive population members, yet salvagers are strongly protected from overexploitation due to partial metabolite privatization. We also describe a previously unnoted benefit of precursor salvaging, namely the removal of the non-functional, proliferation-inhibiting precursor. As long as compatible precursors are present, any microbe possessing the terminal steps of a biosynthetic process can, in principle, forgo de novo biosynthesis in favor of salvaging. Consequently, precursor salvaging likely represents a potent, yet overlooked, alternative to de novo biosynthesis for the acquisition and provisioning of metabolites in free-living bacterial populations.

microbiology↗