Opposite roles of Rad5 in DNA damage tolerance: playing in both error-free and mutagenic lesion bypass

DNA Damage Tolerance (DDT) funcPons to bypass replicaPon-blocking lesions and is divided into two disPnct pathways: error-prone Translesion Synthesis (TLS) and error-free Damage Avoidance (DA). Rad5 is an important player in these processes. Indeed, Saccharomyces cerevisiae Rad5 is a large mulPfuncPonal protein that contains three well defined domains: a RING domain that promotes PCNA polyubiquiPnaPon and a ssDNA-dependent ATPase/helicase domain, that are both conserved in Rad5 human ortholog HLTF. Yeast Rad5 also contains a Rev1-binding domain. In this study we used domain-specific mutants to address the contribuPon of each of the Rad5 funcPons to lesion tolerance. Using an assay based on the inserPon of a single lesion into a defined locus in the genome of a living yeast cell, we demonstrate that Rad5 plays opposite roles in lesion tolerance: i) Rad5 favors error-free lesion bypass by acPvaPng template switching through polyubiquiPnaPon of PCNA; ii) Rad5 is also required for TLS by recruiPng the TLS polymerase Rev1. We also show that the helicase acPvity does not play any role in lesion tolerance/

Snazarus and its human ortholog SNX25 modulate autophagic flux

Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve our understanding of their functions in vivo, we screened all Drosophila SNXs using inducible RNA interference in the fat body. Significantly, depletion of snazarus (snz) led to decreased autophagic flux. Interestingly, we observed altered distribution of Vamp7-positive vesicles with snz depletion, and snz's roles were conserved in human cells. SNX25, the closest human ortholog to snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, we demonstrate that these activities are dependent on specific SNX25 domains and that the autophagic defects upon SNX25 loss can be rescued by ethanolamine addition. We also demonstrate the presence of differentially spliced forms of SNX14 and SNX25 in cancer cells. This work identifies a conserved role for snz/SNX25 as regulators of autophagic flux and reveals differential isoform expression between paralogs.

Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys

In mice, ZP3r/sp56 is a binding partner to the egg coat protein ZP3 and may mediate induction of the acrosome reaction. ZP3r, as a member of the RCA cluster, is surrounded by paralogs, some of which have been shown to be evolving under positive selection. Sequence divergence paired with paralogous relationships with neighboring genes, has complicated the accurate identification of the human ZP3r ortholog. Here, we phylogenetically and syntenically resolve that the human ortholog of ZP3r is the pseudogene C4BPAP1. We investigate the evolution of this gene within primates. We observe independent pseudogenization events of ZP3r in all Apes with the exception of Orangutans, and many monkey species. ZP3r in both primates that retain ZP3r and rodents contains positively selected sites. We hypothesize that redundant mechanisms mediate ZP3 recognition in mammals and ZP3rs relative importance to ZP recognition varies across species.

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition

The mouse 3′-5′ exoribonuclease EXOSC10/Rrp6 is required for rRNA processing, gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cancer cells and its enzymatic activity is inhibited by the anti-cancer drug 5-fluorouracil. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10-/- mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal mRNA is present in mutant oocytes and that the gene is expressed during early embryogenesis. Furthermore, we observe that EXOSC10 localizes to the periphery of nucleolar precursor bodies and nucleoli in blastomeres, which is consistent with the protein's role in rRNA processing. Finally, we infer from genotyping data obtained with samples harvested at embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10-/- mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

Characterization of novel competitive inhibitors of P. falciparum cGMP-dependent protein kinase

P. falciparum cGMP-dependent protein kinase (PfPKG) is an enticing anti-malarial drug target. Structurally novel isoxazole-based compounds were shown to be ATP competitive inhibitors of PfPKG. Isoxazoles 3 and 5 had Ki values of 0.7 (SE = 0.2) and 2.3 (SE = 0.9) nM, respectively, that are comparable to a known standard, 4 [2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (1.4 (SE = 0.5 nM)). They also exhibited excellent selectivity for PfPKG over the human ortholog and the gatekeeper mutant T618Q PfPKG, which mimics the less accessible binding site of the human ortholog. The human ortholog's larger binding site volume was predicted to explain the selectivity of the inhibitors for the P. falciparum enzyme. Analogs 4 and 6 were at least 20-fold less potent compared to 3 and 5, suggesting that removing the carbonyl group in 3 or altering the diethylamino moiety in 5 reduced affinity.

Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-arrestin Recruitment and Receptor Internalization

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2’s NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.

Proteomics of protein trafficking by in vivo tissue-specific labeling

Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

To achieve precise cellular regulation, E3 ubiquitin ligases must be configured to match substrate quaternary structures. Here, by studying the yeast GID complex, mutation of which is Glucose-Induced Degradation deficient, we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryo EM structures show that to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two otherwise functional GID E3s assemble into a 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons and positions Fbp1 so that its protomers are simultaneously ubiquitylated at lysines near its allosteric and substrate binding sites. Significantly, key structural and biochemical features - including capacity for supramolecular assembly - are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical and cell biological data, we propose that higher-order E3 ligase assembly generally underlies multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

Snazarus and its human ortholog SNX25 regulate autophagic flux by affecting VAMP8 endocytosis

Autophagy, the degradation and recycling of cytosolic components in the lysosome, is an essential cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs can impact autophagy. To gain a better understanding of their functions in vivo under nutrient starvation, we screened all Drosophila SNXs by RNAi in the fat body. Significantly, depletion of snazarus (snz) strongly impacted autolysosome formation and led to decreased autophagic flux. Interestingly, we observed altered distribution of Vamp7-positive vesicles with snz depletion and snz roles were conserved in human cells. SNX25 is the closest ortholog to snz, and we demonstrate a role for it in VAMP8 trafficking. We found that this activity was dependent on the SNX25 PX domain, and independent of SNX25 anchoring at the ER. We also demonstrate that differentially spliced forms of SNX14 and SNX25 are present in cancer cells. This work identifies a conserved role for snz/SNX25 as regulators of autophagic flux, and show differential isoform expression between orthologs.

Structural and kinetic characterization of Porphyromonas gingivalis glutaminyl cyclase

Porphyromonas gingivalis is a bacterial species known to be involved in the pathogenesis of chronic periodontitis, that more recently has been as well associated with Alzheimer’s disease. P. gingivalis expresses a glutaminyl cyclase (PgQC) whose human ortholog is known to participate in the beta amyloid peptide metabolism. We have elucidated the crystal structure of PgQC at 1.95 Å resolution in unbound and in inhibitor-complexed forms. The structural characterization of PgQC confirmed that PgQC displays a mammalian fold rather than a bacterial fold. Our biochemical characterization indicates that PgQC uses a mammalian-like catalytic mechanism enabled by the residues Asp149, Glu182, Asp183, Asp218, Asp267 and His299. In addition, we could observe that a non-conserved Trp193 may drive differences in the binding affinity of ligands which might be useful for drug development. With a screening of a small molecule library, we have identified a benzimidazole derivative rendering PgQC inhibition in the low micromolar range that might be amenable for further medicinal chemistry development.