Adipocyte-Derived Exosomal MTTP Suppresses Ferroptosis and Promotes Chemoresistance in Colorectal Cancer.

Obesity is closely related to a poor prognosis in patients with advanced colorectal cancer (CRC), but the mechanisms remain unclear. Ferroptosis is a form of nonapoptotic cell death characterized by lipid reactive oxygen species (ROS) accumulation and iron dependency and is associated with the chemoresistance of tumors. Here, it is shown that adipose-derived exosomes reduce ferroptosis susceptibility in CRC, thus promoting chemoresistance to oxaliplatin. It is found that microsomal triglyceride transfer protein (MTTP) expression is increased in the plasma exosomes of CRC patients with a high body fat ratio, serving as an inhibitor of ferroptosis and reducing sensitivity to chemotherapy. Mechanistically, the MTTP/proline-rich acidic protein 1 (PRAP1) complex inhibited zinc finger E-box binding homeobox 1 expression and upregulated glutathione peroxidase 4 and xCT, leading to a decreased polyunsaturated fatty acids ratio and lipid ROS levels. Moreover, experiments are carried out in organoids, and a tumor implantation model is established in obese mice, demonstrating that the inhibition of MTTP increases the sensitivity to chemotherapy. The results reveal a novel intracellular signaling pathway mediated by adipose-derived exosomes and suggest that treatments targeting secreted MTTP might reverse oxaliplatin resistance in CRC.

Rhamnose-Containing Compounds: Biosynthesis and Applications.

Rhamnose-associated molecules are attracting attention because they are present in bacteria but not mammals, making them potentially useful as antibacterial agents. Additionally, they are also valuable for tumor immunotherapy. Thus, studies on the functions and biosynthetic pathways of rhamnose-containing compounds are in progress. In this paper, studies on the biosynthetic pathways of three rhamnose donors, i.e., deoxythymidinediphosphate-L-rhamnose (dTDP-Rha), uridine diphosphate-rhamnose (UDP-Rha), and guanosine diphosphate rhamnose (GDP-Rha), are firstly reviewed, together with the functions and crystal structures of those associated enzymes. Among them, dTDP-Rha is the most common rhamnose donor, and four enzymes, including glucose-1-phosphate thymidylyltransferase RmlA, dTDP-Glc-4,6-dehydratase RmlB, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase RmlC, and dTDP-4-keto-Rha reductase RmlD, are involved in its biosynthesis. Secondly, several known rhamnosyltransferases from Geobacillus stearothermophilus, Saccharopolyspora spinosa, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Streptococcus pneumoniae are discussed. In these studies, however, the functions of rhamnosyltransferases were verified by employing gene knockout and radiolabeled substrates, which were almost impossible to obtain and characterize the products of enzymatic reactions. Finally, the application of rhamnose-containing compounds in disease treatments is briefly described.

Suppression of DNA Polymerase ß Activity Is Synthetically Lethal in BRCA1-Deficient Cells.

People whose cells express mutated forms of the BRCA1 tumor suppressor are at a higher risk for developing cancer. BRCA1-deficient cells are defective in DNA double-strand break repair. The inhibition of poly(ADP-ribose) polymerase 1 in such cells is a synthetically lethal, cytotoxic effect that has been exploited to produce anticancer drugs such as Olaparib. However, alternative synthetic lethal approaches are necessary. We report that DNA polymerase ß (Pol ß) forms a synthetically lethal interaction with BRCA1. The SiRNA knockdown of Pol ß or the treatment with a Pol ß pro-inhibitor (pro-1) is cytotoxic in BRCA1-deficient ovarian cancer cells. BRCA1-complemented cells are significantly less susceptible to either treatment. pro-1 is also toxic to BRCA1-deficient breast cancer cells, and its toxicity in BRCA1-deficient cells is comparable to that of Olaparib. These experiments establish Pol ß as a synthetically lethal target within BRCA1-deficient cells and a potentially useful one for treating cancer.

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-D-glucose epimerization in Pseudomonas putida KT2440.

L-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-ß-L-rhamnose (dTDP-L-Rha). dTDP-L-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-L-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-L-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-keto-6-deoxy-L-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-L-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

Thymidylate synthase is essential for efficient HIV-1 replication in macrophages.

Thymidylate synthase (TS) is a key enzyme in nucleotide biosynthesis. A study performed by our group on human monocyte-derived macrophages (MDMs) infected with HIV-1 showed that many enzymes related to the folate cycle pathway, such as TS, are upregulated in productively infected cells. Here, we suggest that TS is essential for an effective HIV-1 infection in MDMs. Indeed, a TS specific small interfering RNA (siRNA) as well as the TS specific inhibitor Raltitrexed (RTX) caused a reduction in productively infected cells. Quantitative PCR analysis showed that this treatment decreased the efficacy of the early steps of the viral cycle. The RTX inhibitory effect was counteracted by dNTP addition. These results suggest that TS is essential for the early stages of HIV-1 infection by providing optimal dNTP concentrations in MDMs. TS and its related pathway may thus be considered as a potential therapeutic target for HIV-1 treatment.

1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins.

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA.

The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Šcryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

A strong preference for the TA/TA dinucleotide step discovered for an acridine-based, potent antitumor dsDNA intercalator, C-1305: NMR-driven structural and sequence-specificity studies.

Triazoloacridinone C-1305, a potent antitumor agent recommended for Phase I clinical trials, exhibits high activity towards a wide range of experimental colon carcinomas, in many cases associated with complete tumor regression. C-1305 is a well-established dsDNA intercalator, yet no information on its mode of binding into DNA is available to date. Herein, we present the NMR-driven and MD-refined reconstruction of the 3D structures of the d(CGATATCG)2:C-1305 and d(CCCTAGGG)2:C-1305 non-covalent adducts. In both cases, the ligand intercalates at the TA/TA site, forming well-defined dsDNA:drug 1:1 mol/mol complexes. Orientation of the ligand within the binding site was unambiguously established by the DNA/ligand proton-proton NOE contacts. A subsequent, NMR-driven study of the sequence-specificity of C-1305 using a series of DNA duplexes, allowed us to confirm a strong preference towards TA/TA dinucleotide steps, followed by the TG/CA steps. Interestingly, no interaction at all was observed with duplexes containing exclusively the AT/AT, GG/CC and GA/TC steps.

Quantitation of deoxynucleoside triphosphates by click reactions.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.

DCTPP1 prevents a mutator phenotype through the modulation of dCTP, dTTP and dUTP pools.

To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5'-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity.

Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio.

The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation. Here, we found that Met7-deficient strains show elevated mutation rates, but also increased levels of endogenous DNA damage resulting in gross chromosomal rearrangements (GCRs). Quantification of deoxyribonucleotide (dNTP) pools in cell extracts from met7Δ mutant revealed reductions in dTTP and dGTP that cause a constitutively active DNA damage checkpoint. In addition, we found that the absence of Met7 leads to dUTP accumulation, at levels that allowed its detection in yeast extracts for the first time. Consequently, a high dUTP/dTTP ratio promotes uracil incorporation into DNA, followed by futile repair cycles that compromise both mitochondrial and nuclear DNA integrity. In summary, this work highlights the importance of folate polyglutamylation in the maintenance of nucleotide homeostasis and genome stability.

New detection method for nucleoside triphosphates based on carbon dots: The distance-dependent singlet oxygen trapping.

The distance-dependent based sensing mechanism, such as fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) absorption of gold nanoparticles, has been used widely in visual detection. In this work, we report another distance-dependent detection method for nucleoside triphosphates (NTPs) based on carbon dots (CDs) (1O2 donor) and 9, 10-diphenylanthracene-2-boronic acid (DABA, 1O2 acceptor). The CDs can generate singlet oxygen (1O2) which allows diffusion within 200 nm. Thus, the distance between CDs and DABA decreased through binding of NTPs (<200 nm), leading to absorption changes of DABA under light irradiation due to 1O2 trapping. This sensing system (CDs@DABA) has high selectivity for the detection of NTPs due to the double molecular recognition and a linear response in the 0-80 µM concentration range was accomplished with the detection limit as low as 4.35 µM.

Enzymatic Synthesis of the C-Glycosidic Moiety of Nogalamycin R.

Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C-C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.

Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator.

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.

Mechanistic Implications of the Deamination of TDP-4-amino-4-deoxy-d-fucose Catalyzed by the Radical SAM Enzyme DesII.

DesII is a radical SAM lyase that catalyzes a deamination reaction during the biosynthesis of desosamine in Streptomyces venezuelae. Competing mechanistic hypotheses for this radical-mediated reaction are differentiated according to whether a 1,2-migration takes place and the timing of proton abstraction following generation of a substrate α-hydroxyalkyl radical intermediate. In this study, the deuterated C4 epimer of the natural substrate, TDP-4-amino-4-deoxy-d-[3-2H]fucose, was prepared and shown to be a substrate for DesII undergoing deamination alone with a specific activity that is only marginally reduced (∼3-fold) with respect to that of deamination of the natural substrate. Furthermore, pH titration of the deamination reaction implicates the presence of a hydron acceptor that facilitates catalysis but does not appear to be necessary. On the basis of these as well as previously reported results, a mechanism involving direct elimination of ammonium with concerted proton transfer to the nucleofuge from the adjacent α-hydroxyalkyl radical is proposed.

Whole-genome sequencing reveals principles of brain retrotransposition in neurodevelopmental disorders.

Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are "non-classical" in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We show that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as "lightning rods" for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.

Mitochondrial translation requires folate-dependent tRNA methylation.

Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.

Metformin-induced alterations in nucleotide metabolism cause 5-fluorouracil resistance but gemcitabine susceptibility in oesophageal squamous cell carcinoma.

5-Fluorouracil (5-FU) is a chemotherapeutic agent used to treat a variety of gastric cancers including oesophageal squamous cell carcinoma (OSCC), for which the 5-year mortality rate exceeds 85%. Our study investigated the effects of metformin, an antidiabetic drug with established anti-cancer activity, in combination with 5-FU as a novel chemotherapy strategy, using the OSCC cell lines, WHCO1 and WHCO5. Our results indicate that metformin treatment induces significant resistance to 5-FU in WHCO1 and WHCO5 cells, by more than five- and sixfolds, respectively, as assessed by MTT assay. We show that this is due to global alterations in nucleotide metabolism, including elevated expression of thymidylate synthase and thymidine kinase 1 (established 5-FU resistance mechanisms), which likely result in an increase in intracellular dTTP pools and a "dilution" of 5-FU anabolites. Metformin treatment also increases deoxycytidine kinase (dCK) expression and, as the chemotherapeutic agent gemcitabine relies on dCK for its efficient activity, we speculated that metformin would enhance the sensitivity of OSCC cells to gemcitabine. Indeed we show that metformin pre-treatment greatly increases gemcitabine toxicity and DNA fragmentation in comparison to gemcitabine alone. Taken together, our findings show that metformin alters nucleotide metabolism in OSCC cells and while responsible for inducing resistance to 5-FU, it conversely increases sensitivity to gemcitabine, thereby highlighting metformin and gemcitabine as a potentially novel combination therapy for OSCC.

DNA Polymerase ß Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ß-γ Bridging Group of the Incoming dNTP.

The hydrophobic hinge region of DNA polymerase ß (pol ß) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for the selectivity of the enzyme. Various cancer-associated pol ß variants in the hinge region have reduced fidelity resulting from a decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol ß, has been shown to have a reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a toolkit comprising dNTP bisphosphonate analogues modified at the ß-γ bridging methylene to modulate leaving group (pCXYp mimicking PPi) basicity. Construction of linear free energy relationship plots for the dependence of log(kpol) on leaving group pKa4 revealed that I260M catalyzes dNMP incorporation with a marked negative dependence on leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.

Multifunctional human serum albumin-therapeutic nucleotide conjugate with redox and pH-sensitive drug release mechanism for cancer theranostics.

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2'-deoxyuridine 5'-monophosphate (pTFT). The conjugate allows for direct optical and 19F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.