Cytidine deaminases APOBEC3C and APOBEC3D promote DNA replication stress resistance in pancreatic cancer cells.

Gemcitabine is a potent inhibitor of DNA replication and is a mainstay therapeutic for diverse cancers, particularly pancreatic ductal adenocarcinoma (PDAC). However, most tumors remain refractory to gemcitabine therapies. Here, to define the cancer cell response to gemcitabine, we performed genome-scale CRISPR-Cas9 chemical-genetic screens in PDAC cells and found selective loss of cell fitness upon disruption of the cytidine deaminases APOBEC3C and APOBEC3D. Following gemcitabine treatment, APOBEC3C and APOBEC3D promote DNA replication stress resistance and cell survival by deaminating cytidines in the nuclear genome to ensure DNA replication fork restart and repair in PDAC cells. We provide evidence that the chemical-genetic interaction between APOBEC3C or APOBEC3D and gemcitabine is absent in nontransformed cells but is recapitulated across different PDAC cell lines, in PDAC organoids and in PDAC xenografts. Thus, we uncover roles for APOBEC3C and APOBEC3D in DNA replication stress resistance and offer plausible targets for improving gemcitabine-based therapies for PDAC.

Sex differences in kidney metabolism may reflect sex-dependent outcomes in human diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.

Computational Expansion of High-Resolution-MSn Spectral Libraries.

Commonly, in MS-based untargeted metabolomics, some metabolites cannot be confidently identified due to ambiguities in resolving isobars and structurally similar species. To address this, analytical techniques beyond traditional MS2 analysis, such as MSn fragmentation, can be applied to probe metabolites for additional structural information. In MSn fragmentation, recursive cycles of activation are applied to fragment ions originating from the same precursor ion detected on an MS1 spectrum. This resonant-type collision-activated dissociation (CAD) can yield information that cannot be ascertained from MS2 spectra alone, which helps improve the performance of metabolite identification workflows. However, most approaches for metabolite identification require mass-to-charge (m/z) values measured with high resolution, as this enables the determination of accurate mass values. Unfortunately, high-resolution-MSn spectra are relatively rare in spectral libraries. Here, we describe a computational approach to generate a database of high-resolution-MSn spectra by converting existing low-resolution-MSn spectra using complementary high-resolution-MS2 spectra generated by beam-type CAD. Using this method, we have generated a database, derived from the NIST20 MS/MS database, of MSn spectral trees representing 9637 compounds and 19386 precursor ions where at least 90% of signal intensity was converted from low-to-high resolution.

Protocol for mapping the metabolome and lipidome of medulloblastoma cells using liquid chromatography-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and lipidomics have recently been used to show that MYC-amplified group 3 medulloblastoma tumors are driven by metabolic reprogramming. Here, we present a protocol to extract metabolites and lipids from human medulloblastoma brain tumor-initiating cells and normal neural stem cells. We describe untargeted LC-MS methods that can be used to achieve extensive coverage of the polar metabolome and lipidome. Finally, we detail strategies for metabolite identification and data analysis. For complete details on the use and execution of this protocol, please refer to Gwynne et al.1.

Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma.

MYC-driven medulloblastoma (MB) is an aggressive pediatric brain tumor characterized by therapy resistance and disease recurrence. Here, we integrated data from unbiased genetic screening and metabolomic profiling to identify multiple cancer-selective metabolic vulnerabilities in MYC-driven MB tumor cells, which are amenable to therapeutic targeting. Among these targets, dihydroorotate dehydrogenase (DHODH), an enzyme that catalyzes de novo pyrimidine biosynthesis, emerged as a favorable candidate for therapeutic targeting. Mechanistically, DHODH inhibition acts on target, leading to uridine metabolite scarcity and hyperlipidemia, accompanied by reduced protein O-GlcNAcylation and c-Myc degradation. Pyrimidine starvation evokes a metabolic stress response that leads to cell-cycle arrest and apoptosis. We further show that an orally available small-molecule DHODH inhibitor demonstrates potent mono-therapeutic efficacy against patient-derived MB xenografts in vivo. The reprogramming of pyrimidine metabolism in MYC-driven medulloblastoma represents an unappreciated therapeutic strategy and a potential new class of treatments with stronger cancer selectivity and fewer neurotoxic sequelae.

Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli.

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/ß domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most expanded effector family in the pathogenic arsenal of E. coli.

Galectin-9 binds IgM-BCR to regulate B cell signaling.

The galectin family of secreted lectins have emerged as important regulators of immune cell function; however, their role in B-cell responses is poorly understood. Here we identify IgM-BCR as a ligand for galectin-9. Furthermore, we show enhanced BCR microcluster formation and signaling in galectin-9-deficient B cells. Notably, treatment with exogenous recombinant galectin-9 nearly completely abolishes BCR signaling. We investigated the molecular mechanism for galectin-9-mediated inhibition of BCR signaling using super-resolution imaging and single-particle tracking. We show that galectin-9 merges pre-existing nanoclusters of IgM-BCR, immobilizes IgM-BCR, and relocalizes IgM-BCR together with the inhibitory molecules CD45 and CD22. In resting naive cells, we use dual-color super-resolution imaging to demonstrate that galectin-9 mediates the close association of IgM and CD22, and propose that the loss of this association provides a mechanism for enhanced activation of galectin-9-deficient B cells.

Discovery of Ubiquitin Deamidases in the Pathogenic Arsenal of Legionella pneumophila.

Legionella pneumophila translocates the largest known arsenal of over 330 pathogenic factors, called "effectors," into host cells during infection, enabling L. pneumophila to establish a replicative niche inside diverse amebas and human macrophages. Here, we reveal that the L. pneumophila effectors MavC (Lpg2147) and MvcA (Lpg2148) are structural homologs of cycle inhibiting factor (Cif) effectors and that the adjacent gene, lpg2149, produces a protein that directly inhibits their activity. In contrast to canonical Cifs, both MavC and MvcA contain an insertion domain and deamidate the residue Gln40 of ubiquitin but not Gln40 of NEDD8. MavC and MvcA are functionally diverse, with only MavC interacting with the human E2-conjugating enzyme UBE2N (Ubc13). MavC deamidates the UBE2N∼Ub conjugate, disrupting Lys63 ubiquitination and dampening NF-κB signaling. Combined, our data reveal a molecular mechanism of host manipulation by pathogenic bacteria and highlight the complex regulatory mechanisms integral to L. pneumophila's pathogenic strategy.

The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways.

Host colonization by Gram-negative pathogens often involves delivery of bacterial proteins called "effectors" into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins are the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase-hydrolase domain. Ceg4 could dephosphorylate a broad range of phosphotyrosine-containing peptides in vitro and attenuated activation of MAPK-controlled pathways in both yeast and human cells. Our findings indicate that L. pneumophila's infectious program includes manipulation of phosphorylation cascades in key host pathways. The structural and functional features of the Ceg4 effector unraveled here provide first insight into its function as a phosphotyrosine phosphatase, paving the way to further studies into L. pneumophila pathogenicity.

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

Structural and Functional Survey of Environmental Aminoglycoside Acetyltransferases Reveals Functionality of Resistance Enzymes.

Aminoglycoside N-acetyltransferases (AACs) confer resistance against the clinical use of aminoglycoside antibiotics. The origin of AACs can be traced to environmental microbial species representing a vast reservoir for new and emerging resistance enzymes, which are currently undercharacterized. Here, we performed detailed structural characterization and functional analyses of four metagenomic AAC (meta-AACs) enzymes recently identified in a survey of agricultural and grassland soil microbiomes ( Forsberg et al. Nature 2014 , 509 , 612 ). These enzymes are new members of the Gcn5-Related-N-Acetyltransferase superfamily and confer resistance to the aminoglycosides gentamicin C, sisomicin, and tobramycin. Moreover, the meta-AAC0020 enzyme demonstrated activity comparable with an AAC(3)-I enzyme that serves as a model AAC enzyme identified in a clinical bacterial isolate. The crystal structure of meta-AAC0020 in complex with sisomicin confirmed an unexpected AAC(6') regiospecificity of this enzyme and revealed a drug binding mechanism distinct from previously characterized AAC(6') enzymes. Together, our data highlights the presence of highly active antibiotic-modifying enzymes in the environmental microbiome and reveals unexpected diversity in substrate specificity. These observations of additional AAC enzymes must be considered in the search for novel aminoglycosides less prone to resistance.

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.

Dehalogenimonas sp. Strain WBC-2 Genome and Identification of Its trans-Dichloroethene Reductive Dehalogenase, TdrA.

The Dehalogenimonas population in a dechlorinating enrichment culture referred to as WBC-2 was previously shown to be responsible for trans-dichloroethene (tDCE) hydrogenolysis to vinyl chloride (VC). In this study, blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzymatic assays and protein identification using liquid chromatography coupled with mass spectrometry (LC-MS/MS) led to the functional characterization of a novel dehalogenase, TdrA. This new reductive dehalogenase (RDase) catalyzes the dechlorination of tDCE to VC. A metagenome of the WBC-2 culture was sequenced, and a complete Dehalogenimonas genome, only the second Dehalogenimonas genome to become publicly available, was closed. The tdrA dehalogenase found within the Dehalogenimonas genome appears to be on a genomic island similar to genomic islands found in Dehalococcoides. TdrA itself is most similar to TceA from Dehalococcoides sp. strain FL2 with 76.4% amino acid pairwise identity. It is likely that the horizontal transfer of rdhA genes is not only a feature of Dehalococcoides but also a feature of other Dehalococcoidia, including Dehalogenimonas. A set of primers was developed to track tdrA in WBC-2 subcultures maintained on different electron acceptors. This newest dehalogenase is an addition to the short list of functionally defined RDases sharing the usual characteristic motifs (including an AB operon, a TAT export sequence, two iron-sulfur clusters, and a corrinoid binding domain), substrate flexibility, and evidence for horizontal gene transfer within the Dehalococcoidia.

Molecular Characterization of LubX: Functional Divergence of the U-Box Fold by Legionella pneumophila.

LubX is part of the large arsenal of effectors in Legionella pneumophila that are translocated into the host cytosol during infection. Despite such unique features as the presence of two U-box motifs and its targeting of another effector SidH, the molecular basis of LubX activity remains poorly understood. Here we show that the N terminus of LubX is able to activate an extended number of ubiquitin-conjugating (E2) enzymes including UBE2W, UBEL6, and all tested members of UBE2D and UBE2E families. Crystal structures of LubX alone and in complex with UBE2D2 revealed drastic molecular diversification between the two U-box domains, with only the N-terminal U-box retaining E2 recognition features typical for its eukaryotic counterparts. Extensive mutagenesis followed by functional screening in a yeast model system captured functionally important LubX residues including Arg121, critical for interactions with SidH. Combined, these data provide a new molecular insight into the function of this unique pathogenic factor.