Biology subjects

Wild, R.

Publications and source records attributed to Wild, R..

8 recordsLinked to original sources

bioRxiv2026

A glycosaminoglycan degrading Polysaccharide Utilization Locus in the oral microbiota species Segatella oris

The human gut microbiota has been explored for the last two decades and studies have uncovered its major role in the degradation of complex glycans from the diet as well as the host tissues. Strikingly, until very recently, no similar investigation has been carried out on the human oral cavity microbiota, which is increasingly recognized has an important parameter in gastrointestinal health. Prevotellaceae are an important and under-investigated family of bacteria in both the gut and the oral cavity microbiota. The oral cavity is particularly rich in glycosaminoglycans which could provide an abundant carbon source for the inhabiting microorganisms, and play a significant role in host-pathogen interactions. Hence, we analyzed Polysaccharide Utilization Loci in oral microbiota species and we identified a glycosaminoglycan-degrading Polysaccharide Utilization Locus in Segatella oris. We produced and characterized the four enzymes, confirmed their ability to degrade glycosaminoglycans, and we focused our efforts on the biochemical and structural characterization of the first reported Prevotellaceae endosulfatase.

biochemistry↗

bioRxiv2026

A Glutamine antagonist-modulated tumor microenvironment unleashes enzalutamides immunotherapeutic effects

Androgen receptor (AR) antagonists, such as enzalutamide, suppress prostate cancer (PCa) cells to achieve temporary therapeutic effects. In addition to tumor cell-autonomous suppressive function, AR antagonists can also potentially exert anti-tumor effects via mitigating cytotoxic T cells exhaustion. However, strategies for effectively harnessing enzalutamides immunotherapeutic effects remain elusive. In studying a recently described glutamine antagonist prodrug (DRP-104) in PCa models, we found that despite the initial response, tumors ultimately became resistant. Intriguingly, compared to the untreated (DRP-104 treatment-naive) tumors, the resistant tumors became highly susceptible to enzalutamide in vivo. Additionally, treating tumors with DRP-104 and enzalutamide simultaneously also yielded superior therapeutic efficacy. We demonstrated that DRP-104 therapy promoted the infiltration of CD8+ T cells as well as regulatory T cells (Treg) in responsive tumors, and the tumor-infiltrating Treg were mostly depleted upon enzalutamide treatments. Mechanistically, we showed that Treg differentiation from mouse CD4+ T cells was attenuated by enzalutamide. We further demonstrated that Treg induction was accompanied by the interaction between AR and aryl hydrocarbon receptor (AhR), the nuclear receptor indispensable for Treg differentiation, in the nuclei of CD4+ T cells, and this interaction was diminished by enzalutamide. In further support of AR signaling in Treg biogenesis, analysis of available gene expression datasets found that AR expression was elevated in Treg when compared to CD4+ T cells in human peripheral blood mononuclear cells (PBMCs). In addition, it was positively correlated with Treg module scores in several human cancer types. Finally, using an anti-GPC3 (Glypican 3) vaccination model, we demonstrated that CD4+ T cells subjected to Treg induction in the presence of enzalutamide were less effective in protecting GPC3-expressing tumor cells from CD8+ T cells cytotoxic killing. Collectively, these results suggest that AR promotes Treg s differentiation and/or immunosuppressive functions, and nominate enzalutamide as a Treg-mitigating agent for potentiating immunotherapies. Our results also demonstrate that an otherwise unintended, Treg-promoting property of DRP-104 can be leveraged to unleash the immune-regulatory function of enzalutamide for the treatment of PCa.

cancer biology↗

bioRxiv2025

Structural basis for human chondroitin sulfate chain polymerization

Chondroitin sulfates are complex polysaccharide chains that regulate various biological processes at the cell surface and within the extracellular matrix. Here, we identify four heterodimeric complexes responsible for chondroitin sulfate chain polymerization in humans: CHSY1-CHPF, CHSY1-CHPF2, CHSY3-CHPF and CHSY3-CHPF2. Using a novel in vitro glycosylation assay based on chemo-enzymatically synthesized fluorescent substrates, we demonstrate that all four complexes exhibit chain polymerization activity. The cryo-EM structure of the CHSY3-CHPF complex provides, for the first time, molecular insights into the chondroitin sulfate chain polymerization reaction. The architecture of the catalytic sites suggests that CHSY1 and CHSY3 are enzymatically active, while CHPF and CHPF2 primarily play a stabilizing role. Mutational analysis of purified enzyme complexes, combined with an in cellulo complementation assay, confirms that only CHSY1 and CHSY3 have bifunctional glycosyltransferase activities. Based on the spatial arrangement of the catalytic sites, we propose that chondroitin sulfates chain polymerization follows a non-processive, disruptive mechanism.

molecular biology↗

bioRxiv2025

Resilience and vulnerabilities of tumor cells under purine shortage stress

Purine metabolism is a promising therapeutic target in cancer; however how cancer cells respond to purine shortage,particularly their adaptation and vulnerabilities, remains unclear. Using the recently developed purine shortage-inducing prodrug DRP-104 and genetic approaches, we investigated these responses in prostate, lung and glioma cancer models. We demonstrate that when de novo purine biosynthesis is compromised, cancer cells employ microtubules to assemble purinosomes, multi-protein complexes of de novo purine biosynthesis enzymes that enhance purine biosynthesis efficiency. While this process enables tumor cells to adapt to purine shortage stress, it also renders them more susceptible to the microtubule-stabilizing chemotherapeutic drug Docetaxel. Furthermore, we show that although cancer cells primarily rely on de novo purine biosynthesis, they also exploit Methylthioadenosine Phosphorylase (MTAP)-mediated purine salvage as a crucial alternative source of purine supply, especially under purine shortage stress. In support of this finding, combining DRP-104 with an MTAP inhibitor significantly enhances tumor suppression in prostate cancer (PCa) models in vivo. Finally, despite the resilience of the purine supply machinery, purine shortage-stressed tumor cells exhibit increased DNA damage and activation of the cGAS-STING pathway, which may contribute to impaired immunoevasion and provide a molecular basis of the previously observed DRP-104-induced anti-tumor immunity. Together, these findings reveal purinosome assembly and purine salvage as key mechanisms of cancer cell adaptation and resilience to purine shortage while identifying microtubules, MTAP, and immunoevasion deficits as therapeutic vulnerabilities.

cancer biology↗

bioRxiv2023

Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.

cancer biology↗

bioRxiv2022

Structure of the human heparan sulfate polymerase complex EXT1-EXT2

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains with their catalytic sites facing in opposite directions. Along with in vitro activity assays using fluorescently labeled and chemically defined substrates, these findings provide a molecular insight into donor substrate recognition and demonstrate that the glycosyltransferase reactions are highly specific. A combination of in vitro and in cellulo mutational studies was used to dissect the functional role of the four catalytic sites. While EXT1 is able to catalyze both glycosyltransferase reactions, EXT2 harbors only N-acetylglucosamine transferase activity. Our results provide mechanistic insight into heparan sulfate chain elongation as a non processive process and lay the cornerstone for future studies on EXT1-EXT2 function in health and disease.

molecular biology↗

bioRxiv2021

Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferases

The oligosaccharyltransferase (OST) is the central enzyme in the N-glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analysed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzyme in vitro and the more severe hypoglycosylation in Ost3p lacking strains in vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition.

biochemistry↗

bioRxiv2019

Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signalling cascades, enabling them to maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signalling molecules (PP-InsPs), which are sensed by SPX-domain containing proteins. In plants, PP-InsP bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8 - SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors. (173 words)

plant biology↗