Chemoproteomic development of SLC15A4 inhibitors with anti-inflammatory activity.

SLC15A4 is an endolysosome-resident transporter linked with autoinflammation and autoimmunity. Specifically, SLC15A4 is critical for Toll-like receptors (TLRs) 7-9 as well as nucleotide-binding oligomerization domain-containing protein (NOD) signaling in several immune cell subsets. Notably, SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential, the availability of quality chemical probes targeting SLC15A4 functions is limited. In this study, we used an integrated chemical proteomics approach to develop a suite of chemical tools, including first-in-class functional inhibitors, for SLC15A4. We demonstrate that these inhibitors suppress SLC15A4-mediated endolysosomal TLR and NOD functions in a variety of human and mouse immune cells; we provide evidence of their ability to suppress inflammation in vivo and in clinical settings; and we provide insights into their mechanism of action. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune and autoinflammatory conditions.

PARP7-mediated ADP-ribosylation of FRA1 promotes cancer cell growth by repressing IRF1- and IRF3-dependent apoptosis.

PARP7 was reported to promote tumor growth in a cell-autonomous manner and by repressing the antitumor immune response. Nevertheless, the molecular mechanism of how PARP7-mediated ADP-ribosylation exerts these effects in cancer cells remains elusive. Here, we identified PARP7 as a nuclear and cysteine-specific mono-ADP-ribosyltransferase that modifies targets critical for regulating transcription, including the AP-1 transcription factor FRA1. Loss of FRA1 ADP-ribosylation via PARP7 inhibition by RBN-2397 or mutation of the ADP-ribosylation site C97 increased FRA1 degradation by the proteasome via PSMC3. The reduction in FRA1 protein levels promoted IRF1- and IRF3-dependent cytokine as well as proapoptotic gene expression, culminating in CASP8-mediated apoptosis. Furthermore, high PARP7 expression was indicative of the PARP7 inhibitor response in FRA1-positive lung and breast cancer cells. Collectively, our findings highlight the connected roles of PARP7 and FRA1 and emphasize the clinical potential of PARP7 inhibitors for FRA1-driven cancers.

Loss of function of ENT3 drives histiocytosis and inflammation through TLR-MAPK signaling.

Histiocytoses are inflammatory myeloid neoplasms often driven by somatic activating mutations in mitogen-activated protein kinase (MAPK) cascade genes. H syndrome is an inflammatory genetic disorder caused by germ line loss-of-function mutations in SLC29A3, encoding the lysosomal equilibrative nucleoside transporter 3 (ENT3). Patients with H syndrome are predisposed to develop histiocytosis, yet the mechanism is unclear. Here, through phenotypic, molecular, and functional analysis of primary cells from a cohort of patients with H syndrome, we reveal the molecular pathway leading to histiocytosis and inflammation in this genetic disorder. We show that loss of function of ENT3 activates nucleoside-sensing toll-like receptors (TLR) and downstream MAPK signaling, inducing cytokine secretion and inflammation. Importantly, MEK inhibitor therapy led to resolution of histiocytosis and inflammation in a patient with H syndrome. These results demonstrate a yet-unrecognized link between a defect in a lysosomal transporter and pathological activation of MAPK signaling, establishing a novel pathway leading to histiocytosis and inflammation.

IFN-stimulated metabolite transporter ENT3 facilitates viral genome release.

An increasing amount of evidence emphasizes the role of metabolic reprogramming in immune cells to fight infections. However, little is known about the regulation of metabolite transporters that facilitate and support metabolic demands. In this study, we found that the expression of equilibrative nucleoside transporter 3 (ENT3, encoded by solute carrier family 29 member 3, Slc29a3) is part of the innate immune response, which is rapidly upregulated upon pathogen invasion. The transcription of Slc29a3 is directly regulated by type I interferon-induced signaling, demonstrating that this metabolite transporter is an interferon-stimulated gene (ISG). Suprisingly, we unveil that several viruses, including SARS-CoV-2, require ENT3 to facilitate their entry into the cytoplasm. The removal or suppression of Slc29a3 expression is sufficient to significantly decrease viral replication in vitro and in vivo. Our study reveals that ENT3 is a pro-viral ISG co-opted by some viruses to gain a survival advantage.

Equilibrative nucleotide transporter ENT3 (SLC29A3): A unique transporter for inherited disorders and cancers.

As a crucial gene associated with diseases, the SLC29A3 gene encodes the equilibrative nucleoside transporter 3 (ENT3). ENT3 plays an essential regulatory role in transporting intracellular hydrophilic nucleosides, nucleotides, hydrophilic anticancer and antiviral nucleoside drugs, energy metabolism, subcellular localization, protein stability, and signal transduction. The mutation and inactivation of SLC29A3 are intimately linked to the occurrence, development, and prognosis of various human tumors. Moreover, many hereditary human diseases, such as H syndrome, pigmentary hypertrichosis and non-autoimmune insulin-dependent diabetes mellitus (PHID) syndrome, Faisalabad histiocytosis (FHC), are related to SLC29A3 mutations. This review explores the mechanisms of SLC29A3 mutations and expression alterations in inherited disorders and cancers. Additionally, we compile studies on the inhibition of ENT3, which may serve as an effective strategy to potentiate the anticancer activity of chemotherapy. Thus, the synopsis of genetics, permeant function and drug therapy of ENT3 provides a new theoretical and empirical foundation for the diagnosis, prognosis of evaluation and treatment of various related diseases.

Transcriptome screening identifies TIPARP as an antiviral host factor against the Getah virus.

IMPORTANCE: Alphaviruses threaten public health continuously, and Getah virus (GETV) is a re-emerging alphavirus that can potentially infect humans. Approved antiviral drugs and vaccines against alphaviruses are few available, but several host antiviral factors have been reported. Here, we used GETV as a model of alphaviruses to screen for additional host factors. Tetrachlorodibenzo-p-dioxin-inducible poly(ADP ribose) polymerase was identified to inhibit GETV replication by inducing ubiquitination of the glycoprotein E2, causing its degradation by recruiting the E3 ubiquitin ligase membrane-associated RING-CH8 (MARCH8). Using GETV as a model virus, focusing on the relationship between viral structural proteins and host factors to screen antiviral host factors provides new insights for antiviral studies on alphaviruses.

A Novel Fluorescent Gemcitabine Prodrug That Follows a Nucleoside Transporter-Independent Internalization and Bears Enhanced Therapeutic Efficacy With Respect to Gemcitabine.

The multiplexity of cancer has rendered it the second leading cause of mortality worldwide and theragnostic prodrugs have gained popularity in recent years as a means of treatment. Theragnostic prodrugs enable the simultaneous diagnosis and therapy of tumors via high-precision real-time drug release monitoring. Herein, we report the development of the small theragnostic prodrug GF, based on the nucleoside anticancer agent gemcitabine and the fluorescent dye 5(6)-carboxyfluorescein. We have successfully demonstrated its efficient internalization in tumor cells, showing localization throughout both the early and late endocytic pathways. Its mechanism of cell internalization was evaluated, confirming its independence from nucleoside transporters. Its cellular localization via confocal microscopy revealed a clathrin-mediated endocytosis mechanism, distinguishing it from analogous compounds studied previously. Furthermore, GF exhibited stability across various pH values and in human blood plasma. Subsequently, its in vitro cytotoxicity was assessed in three human cancer cell lines (A549, U87 and T98). Additionally, its pharmacokinetic profile in mice was investigated and the consequent drug release was monitored. Finally, its in vivo visualization was accomplished in zebrafish xenotransplantation models and its in vivo efficacy was evaluated in A549 xenografts. The results unveiled an intriguing efficacy profile, positioning GF as a compelling candidate warranting further investigation.

Equilibrative nucleoside transporter 3 supports microglial functions and protects against the progression of Huntington's disease in the mouse model.

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by involuntary movements, cognitive deficits, and psychiatric symptoms. Currently, there is no cure, and only limited treatments are available to manage the symptoms and to slow down the disease's progression. The molecular and cellular mechanisms of HD's pathogenesis are complex, involving immune cell activation, altered protein turnover, and disturbance in brain energy homeostasis. Microglia have been known to play a dual role in HD, contributing to neurodegeneration through inflammation but also enacting neuroprotective effects by clearing mHTT aggregates. However, little is known about the contribution of microglial metabolism to HD progression. This study explores the impact of a microglial metabolite transporter, equilibrative nucleoside transporter 3 (ENT3), in HD. Known as a lysosomal membrane transporter protein, ENT3 is highly enriched in microglia, with its expression correlated with HD severity. Using the R6/2 ENT3-/- mouse model, we found that the deletion of ENT3 increases microglia numbers yet worsens HD progression, leading to mHTT accumulation, cell death, and disturbed energy metabolism. These results suggest that the delicate balance between microglial metabolism and function is crucial for maintaining brain homeostasis and that ENT3 has a protective role in ameliorating neurodegenerative processes.

Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods.

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

Toward a Molecular Basis of Cellular Nucleoside Transport in Humans.

Nucleosides play central roles in all facets of life, from metabolism to cellular signaling. Because of their physiochemical properties, nucleosides are lipid bilayer impermeable and thus rely on dedicated transport systems to cross biological membranes. In humans, two unrelated protein families mediate nucleoside membrane transport: the concentrative and equilibrative nucleoside transporter families. The objective of this review is to provide a broad outlook on the current status of nucleoside transport research. We will discuss the role played by nucleoside transporters in human health and disease, with emphasis placed on recent structural advancements that have revealed detailed molecular principles of these important cellular transport systems and exploitable pharmacological features.

Visualizing multistep elevator-like transitions of a nucleoside transporter.

Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.

A semi-synthetic organism that stores and retrieves increased genetic information.

Since at least the last common ancestor of all life on Earth, genetic information has been stored in a four-letter alphabet that is propagated and retrieved by the formation of two base pairs. The central goal of synthetic biology is to create new life forms and functions, and the most general route to this goal is the creation of semi-synthetic organisms whose DNA harbours two additional letters that form a third, unnatural base pair. Previous efforts to generate such semi-synthetic organisms culminated in the creation of a strain of Escherichia coli that, by virtue of a nucleoside triphosphate transporter from Phaeodactylum tricornutum, imports the requisite unnatural triphosphates from its medium and then uses them to replicate a plasmid containing the unnatural base pair dNaM-dTPT3. Although the semi-synthetic organism stores increased information when compared to natural organisms, retrieval of the information requires in vivo transcription of the unnatural base pair into mRNA and tRNA, aminoacylation of the tRNA with a non-canonical amino acid, and efficient participation of the unnatural base pair in decoding at the ribosome. Here we report the in vivo transcription of DNA containing dNaM and dTPT3 into mRNAs with two different unnatural codons and tRNAs with cognate unnatural anticodons, and their efficient decoding at the ribosome to direct the site-specific incorporation of natural or non-canonical amino acids into superfolder green fluorescent protein. The results demonstrate that interactions other than hydrogen bonding can contribute to every step of information storage and retrieval. The resulting semi-synthetic organism both encodes and retrieves increased information and should serve as a platform for the creation of new life forms and functions.

Molecular basis for substrate recognition by the bacterial nucleoside transporter NupG.

Nucleoside homeostasis, which is mediated by transporters and channels, is essential for all life on Earth. In Escherichia coli, NupG mediates the transport of nucleosides and was deemed to be the prototype of the nucleoside proton symporter (NHS) family and the major facilitator superfamily. To date, the substrate recognition and transport mechanisms of NHS transporters are still elusive. Here, we report two crystal structures of NupG (WT and D323A NupG) resolved at 3.0 Å. Both structures reveal an identical inward-open conformation. Together with molecular docking and molecular dynamics simulations and in vitro uridine-binding assays, we found that the uridine binding site, which locates in the central cavity between N and C domains of NupG, is constituted by R136, T140, F143, Q225, N228, Q261, E264, Y318, and F322. Moreover, we found that D323 is very important for substrate binding via in vitro uridine-binding assays using D323 mutations, although it does not have a direct contact with uridine. Our structural and biochemical data therefore provide an important framework for the mechanistic understanding of nucleoside transporters of the NHS family.

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter.

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.

Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters.

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.

PARP7 mono-ADP-ribosylates the agonist conformation of the androgen receptor in the nucleus.

We recently described a signal transduction pathway that contributes to androgen receptor (AR) regulation based on site-specific ADP-ribosylation by PARP7, a mono-ADP-ribosyltransferase implicated in several human cancers. ADP-ribosylated AR is recognized by PARP9/DTX3L, a heterodimeric complex that contains an ADP-ribose reader (PARP9) and a ubiquitin E3 ligase (DTX3L). Here, we have characterized the cellular and biochemical requirements for AR ADP-ribosylation by PARP7. We found that the reaction requires nuclear localization of PARP7 and an agonist-induced conformation of AR. PARP7 contains a Cys3His1-type zinc finger (ZF), which also is critical for AR ADP-ribosylation. The Parp7 ZF is required for efficient nuclear import by a nuclear localization signal encoded in PARP7, but rescue experiments indicate the ZF makes a contribution to AR ADP-ribosylation that is separable from the effect on nuclear transport. ZF mutations do not detectably reduce PARP7 catalytic activity and binding to AR, but they do result in the loss of PARP7 enhancement of AR-dependent transcription of the MYBPC1 gene. Our data reveals critical roles for AR conformation and the PARP7 ZF in AR ADP-ribosylation and AR-dependent transcription.

Inborn Errors of Nucleoside Transporter (NT)-Encoding Genes (SLC28 and SLC29).

The proper regulation of nucleotide pools is essential for all types of cellular functions and depends on de novo nucleotide biosynthesis, salvage, and degradation pathways. Despite the apparent essentiality of these processes, a significant number of rare diseases associated with mutations in genes encoding various enzymes of these pathways have been already identified, and others are likely yet to come. However, knowledge on genetic alterations impacting on nucleoside and nucleobase transporters is still limited. At this moment three gene-encoding nucleoside and nucleobase transporter proteins have been reported to be mutated in humans, SLC29A1, SLC29A3, and SLC28A1, impacting on the expression and function of ENT1, ENT3, and CNT1, respectively. ENT1 alterations determine Augustine-null blood type and cause ectopic calcification during aging. ENT3 deficiency translates into various clinical manifestations and syndromes, altogether listed in the OMIM catalog as histiocytosis-lymphoadenopathy plus syndrome (OMIM#602782). CNT1 deficiency causes uridine-cytidineuria (URCTU) (OMIM#618477), a unique type of pyrimidineuria with an as yet not well-known clinical impact. Increasing knowledge on the physiological, molecular and structural features of these transporter proteins is helping us to better understand the biological basis behind the biochemical and clinical manifestations caused by these deficiencies. Moreover, they also support the view that some metabolic compensation might occur in these disturbances, because they do not seem to significantly impact nucleotide homeostasis, but rather other biological events associated with particular subtypes of transporter proteins.

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer.

Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis despite current best therapies; therefore new treatment strategies are urgently required. Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of PDAC. EMT is associated with phenotypic conversion of epithelial cells into mesenchymal-like cells in cell culture conditions, although such defined mesenchymal conversion (with spindle-shaped morphology) of epithelial cells in vivo is rare, with quasi-mesenchymal phenotypes occasionally observed in the tumour (partial EMT). Most studies exploring the functional role of EMT in tumours have depended on cell-culture-induced loss-of-function and gain-of-function experiments involving EMT-inducing transcription factors such as Twist, Snail and Zeb1 (refs 2, 3, 7-10). Therefore, the functional contribution of EMT to invasion and metastasis remains unclear, and genetically engineered mouse models to address a causal connection are lacking. Here we functionally probe the role of EMT in PDAC by generating mouse models of PDAC with deletion of Snail or Twist, two key transcription factors responsible for EMT. EMT suppression in the primary tumour does not alter the emergence of invasive PDAC, systemic dissemination or metastasis. Suppression of EMT leads to an increase in cancer cell proliferation with enhanced expression of nucleoside transporters in tumours, contributing to enhanced sensitivity to gemcitabine treatment and increased overall survival of mice. Collectively, our study suggests that Snail- or Twist-induced EMT is not rate-limiting for invasion and metastasis, but highlights the importance of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.

Nucleoside transporter-guided cytarabine-conjugated liposomes for intracellular methotrexate delivery and cooperative choriocarcinoma therapy.

Gestational trophoblastic tumors seriously endanger child productive needs and the health of women in childbearing age. Nanodrug-based therapy mediated by transporters provides a novel strategy for the treatment of trophoblastic tumors. Focusing on the overexpression of human equilibrative nucleoside transporter 1 (ENT1) on the membrane of choriocarcinoma cells (JEG-3), cytarabine (Cy, a substrate of ENT1)-grafted liposomes (Cy-Lipo) were introduced for the targeted delivery of methotrexate (Cy-Lipo@MTX) for choriocarcinoma therapy in this study. ENT1 has a high affinity for Cy-Lipo and can mediate the endocytosis of the designed nanovehicles into JEG-3 cells. The ENT1 protein maintains its transportation function through circulation and regeneration during endocytosis. Therefore, Cy-Lipo-based formulations showed high tumor accumulation and retention in biodistribution studies. More importantly, the designed DSPE-PEG2k-Cy conjugation exhibited a synergistic therapeutic effect on choriocarcinoma. Finally, Cy-Lipo@MTX exerted an extremely powerful anti-choriocarcinoma effect with fewer side effects. This study suggests that the overexpressed ENT1 on choriocarcinoma cells holds great potential as a high-efficiency target for the rational design of active targeting nanotherapeutics.