ABSTRACT
In plants, the conserved plant-specific photoreceptor UV RESISTANCE LOCUS 8 (UVR8) perceives ultraviolet-B (UV-B) light and mediates UV-B-induced photomorphogenesis and stress acclimation. In this study, we reveal that UV-B light treatment shortens seedlings, increases stem thickness, and enhances UV-B stress tolerance in rice (Oryza sativa) via its two UV-B photoreceptors OsUVR8a and OsUVR8b. Although the rice and Arabidopsis (Arabidopsis thaliana) UVR8 (AtUVR8) photoreceptors all form monomers in response to UV-B light, OsUVR8a, and OsUVR8b function is only partially conserved with respect to AtUVR8 in UV-B-induced photomorphogenesis and stress acclimation. UV-B light and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) promote the nuclear accumulation of AtUVR8; by contrast, OsUVR8a and OsUVR8b constitutively localize to the nucleus via their own nuclear localization signals, independently of UV-B light and the RING-finger mutation of OsCOP1. We show that OsCOP1 negatively regulates UV-B responses, and shows weak interaction with OsUVR8s, which is ascribed to the N terminus of OsCOP1, which is conserved in several monocots. Furthermore, transcriptome analysis demonstrates that UV-B-responsive gene expression differs globally between Arabidopsis and rice, illuminating the evolutionary divergence of UV-B light signaling pathways between monocot and dicot plants.
Subject(s)
Arabidopsis , Cell Nucleus , Gene Expression Regulation, Plant , Oryza , Plant Proteins , Ultraviolet Rays , Oryza/metabolism , Oryza/genetics , Oryza/radiation effects , Cell Nucleus/metabolism , Cell Nucleus/radiation effects , Gene Expression Regulation, Plant/radiation effects , Plant Proteins/metabolism , Plant Proteins/genetics , Arabidopsis/radiation effects , Arabidopsis/metabolism , Arabidopsis/genetics , Photoreceptors, Plant/metabolism , Photoreceptors, Plant/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Seedlings/radiation effects , Seedlings/metabolism , Seedlings/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Mutation , Plants, Genetically Modified , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/geneticsABSTRACT
VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.
Subject(s)
Autophagosomes , Autophagy , Autophagosomes/metabolism , Autophagy/physiology , Intracellular Membranes/metabolism , Endosomes/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolismABSTRACT
The VPS37A gene encodes a subunit of the endosomal sorting complex required for transport (ESCRT)-I complex that is frequently lost in a wide variety of human solid cancers. We have previously demonstrated the role of VPS37A in directing the ESCRT membrane scission machinery to seal the phagophore for autophagosome completion. Here, we report that VPS37A-deficient cells exhibit an accumulation of the apoptotic initiator CASP8 (caspase 8) on the phagophore and are primed to undergo rapid apoptosis through the intracellular death-inducing signaling complex (iDISC)-mediated CASP8 activation upon exposure to endoplasmic reticulum (ER) stress. Using CRISPR-Cas9 gene editing and comparative transcriptome analysis, we identified the ATF4-mediated stress response pathway as a crucial mediator to elicit iDISC-mediated apoptosis following the inhibition of autophagosome closure. Notably, ATF4-mediated iDISC activation occurred independently of the death receptor TNFRSF10B/DR5 upregulation but required the pro-apoptotic transcriptional factor DDIT3/CHOP to enhance the mitochondrial amplification pathway for full-activation of CASP8 in VPS37A-deficient cells stimulated with ER stress inducers. Our analysis also revealed the upregulation of NFKB/NF-kB signaling as a potential mechanism responsible for restraining iDISC activation and promoting cell survival upon VPS37A depletion. These findings have important implications for the future development of new strategies to treat human cancers, especially those with VPS37A loss.Abbreviations: ATG: autophagy related; BMS: BMS-345541; CASP: caspase; CHMP: charged multivesicular body protein; DKO: double knockout; Dox: doxycycline; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; gRNA: guide RNA; GSEA: gene set enrichment analysis; GSK157: GSK2656157; iDISC: intracellular death-inducing signaling complex; IKK: inhibitor of NFKB kinase; IPA: ingenuity pathway analysis; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NFKB/NF-kB: nuclear factor kappa B; OZ: 5Z-7-oxozeaenol; RNA-seq: RNA sequencing; UPR: unfolded protein response; TFT: transcription factor target; THG: thapsigargin; TUN: tunicamycin; VPS: vacuolar protein sorting.
Subject(s)
NF-kappa B , Neoplasms , Humans , Caspase 8/genetics , NF-kappa B/metabolism , Autophagy , RNA, Guide, CRISPR-Cas Systems , Apoptosis/genetics , Endoplasmic Reticulum Stress , Endosomal Sorting Complexes Required for Transport/metabolismABSTRACT
Using Gallic acid as raw material, 1-(substituted aromatic acyl)-4-(3,4,5-trihydroxybenzoyl) thiosemicarbazone was prepared by a two-step reaction and a series of brand-new gallic acid amide derivatives that contained 1,3,4-thiadiazole were synthesized by cyclic reaction. The newly prepared compounds' Vibrio harveyi inhibition activities were evaluated. The results indicated that all compounds showed different degree of inhibitory activity on Vibrio harveyi. Among them, the best inhibition effect was shown by compound 5b and its minimum inhibitory concentration (MIC) was 0.0313mg/mL.
Subject(s)
Gallic Acid , Vibrio , Gallic Acid/pharmacology , Amides/pharmacologyABSTRACT
Listeria monocytogenes is an important foodborne pathogen worldwide, with 20-30% fatality rate in vulnerable persons. The hypervirulent L. monocytogenes clonal complex (CC) 87 strains have emerging both in food production environments and clinic cases. The objective of this study was to develop a multiplex PCR to simultaneously detect L. monocytogenes CC87 and CC88 strains based on pan-genome analysis. A novel multiplex PCR comprised of genes A6K41_13255 (specific for CC87 and 88), BCW_4260_01987 group_8135 (specific for CC88) and 02-1103_01073 group_5869 (specific for L. monocytogenes) were designed. The specificity of this multiplex PCR was robust verified with other CCs of L. monocytogenes and other species strains. The detection limit of this multiplex PCR for CC87 and CC88 were 1.7 × 104 cfu/mL and 2.1 × 104 cfu/mL, respectively. This multiplex PCR could accurately detect CC87 and CC88 strains with the interference of different ratios of L. monocytogenes CC8, CC9, CC121, CC155, and L. innocua strains. Furthermore, this multiplex PCR method could successfully detect 1.9 × 104 cfu/mL of L. monocytogenes CC87 and 1.7 × 104 cfu/mL CC88 strains in artificially contaminated milk after 9 h enrichment, respectively. In addition, this multiplex PCR could accurately detect CC87 isolates in food samples within 48 h, which was faster than the routine MLST analysis. In conclusion, this novel multiplex PCR offers a promising approach for accurate, inexpensive, and rapid detection of L. monocytogenes CC87 and CC88 strains simultaneously, which could apply to surveillance the prevalence of CC87 and CC88 strains in both food and food production environments and to evaluate the effect of disinfection measures for controlling the persistent L. monocytogenes contamination.
Subject(s)
Listeria monocytogenes , Animals , Food Microbiology , Milk , Multilocus Sequence Typing , Multiplex Polymerase Chain ReactionABSTRACT
Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of l-proline biosynthesis by catalyzing the reduction of L-Δ1-pyrroline-5-carboxylate (L-P5C) to l-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology.
Subject(s)
Disease/genetics , Mutation , Pyrroline Carboxylate Reductases/genetics , Enzyme Stability , Humans , Kinetics , Protein Structure, Secondary , Pyrroline Carboxylate Reductases/chemistry , Pyrroline Carboxylate Reductases/metabolism , TemperatureABSTRACT
Autophagosomal membranes can serve as activation platforms for intracellular death-inducing signaling complexes (iDISCs) to initiate Caspase-8-dependent apoptosis. In this study, we explore the impact of ESCRT-III-dependent phagophore closure on iDISC assemblies and cell death in osteosarcoma and neuroblastoma cells. Inhibition of phagophore closure by conditional depletion of CHMP2A, an ESCRT-III component, stabilizes iDISCs on immature autophagosomal membranes and induces Caspase-8-dependent cell death. Importantly, suppression of the iDISC formation via deletion of ATG7, an E1 enzyme for ubiquitin-like autophagy-related proteins, blocks Caspase-8 activation and cell death following CHMP2A depletion. Although DR5 expression and TRAIL-induced apoptosis are enhanced in CHMP2A-depleted cells, the canonical extrinsic pathway of apoptosis is not responsible for the initiation of cell death by CHMP2A depletion. Furthermore, the loss of CHMP2A impairs neuroblastoma tumor growth associated with decreased autophagy and increased apoptosis in vivo. Together, these findings indicate that inhibition of the ESCRT-III-dependent autophagosome sealing process triggers noncanonical Caspase-8 activation and apoptosis, which may open new avenues for therapeutic targeting of autophagy in cancer.
Subject(s)
Autophagy , Caspase 8/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Osteosarcoma/metabolism , Signal Transduction , Animals , Apoptosis , Autophagosomes/metabolism , Cell Line, Tumor , Endosomal Sorting Complexes Required for Transport/genetics , Female , Humans , Male , Mice , Neuroblastoma/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Poly(ADP-ribose) Polymerase Inhibitors/isolation & purification , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/drug effects , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Biomarkers , DNA Damage , DNA Repair , Gene Knockout Techniques , HeLa Cells , Homologous Recombination/drug effects , Humans , Lysine Acetyltransferase 5/metabolism , Mad2 Proteins/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1 , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, multivesicular body biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101-VPS28-VPS37B-MVB12A was determined, revealing an ESCRT-I helical assembly with a 12-molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse-grained (CG) simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor; it has an essential scaffolding and mechanical role in its own right.
Subject(s)
Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/metabolism , HIV-1/physiology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , Autophagosomes , Cell Membrane/metabolism , Crystallization , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , HEK293 Cells/virology , HIV-1/metabolism , Host-Pathogen Interactions/physiology , Humans , Molecular Dynamics Simulation , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Protein Conformation , Transcription Factors/chemistry , Transcription Factors/metabolism , Virus Release , gag Gene Products, Human Immunodeficiency Virus/chemistry , gag Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Bee pollen (BP) shows profound gut-protecting potentials. BP lipids (BPLs) mainly composed by phospholipids and polyunsaturated fatty acids might be one of the important contributors, while how BPL exerts gut-protecting effects and is transported through intestinal cell monolayers need to be investigated. Here, we exploited a strategy that combines an UPLC-Q-exactive orbitrap/MS-based lipidomics approach with a human intestinal cell (Caco-2) monolayer transport model, to determine the transepithelial transportation of BPL from Camellia sinensis L. (BPL-Cs), in pathological conditions. The results showed that BPL-Cs protected Caco-2 cells against dextran sulfate sodium (DSS)-induced intestinal barrier dysfunction by improving cell viability, maintaining membrane integrity, increasing tight junctions (ZO-1 and Claudin-1), and eliciting the expressions of antioxidative-related genes (NQO1, Nrf2, Txnrd1, and GSTA1). Lipidomics analysis revealed that DSS suppressed the transport and uptake of most of BPL-Cs including glycerophospholipids, sphingomyelins, and glycosylsphingolipids. Pretreatment with BPL-Cs significantly regulated glycerophospholipid and sphingolipid metabolisms, potentially involved in building permeability barriers and alleviating intestinal oxidative stress. Finally, eight classes of lipids were identified as the potential biomarkers for evaluating DSS-induced Caco-2 cell dysfunctions and BPL-intervened modulation. These findings shed light on the development of BPL as gastrointestinal protective food supplements in the future.
Subject(s)
Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , Lipid Metabolism , Pollen/metabolism , Animals , Bees , Biological Transport , Caco-2 Cells , Camellia sinensis/chemistry , Claudin-1/genetics , Claudin-1/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Lipids/chemistry , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Pollen/chemistryABSTRACT
The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Phagosomes/metabolism , Cells, Cultured , HEK293 Cells , HeLa Cells , HumansABSTRACT
During autophagy, phagophores grow into double-membrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or non-vesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.
Subject(s)
Autophagosomes/metabolism , Autophagy-Related Proteins/metabolism , Autophagy , Endoplasmic Reticulum/metabolism , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Amino Acid Sequence , Autophagy-Related Proteins/genetics , HEK293 Cells , Humans , Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Phosphatidylinositol Phosphates/metabolism , Sequence HomologyABSTRACT
Bee pollen (BP) is a natural medicine from the hive with various potential health-promoting benefits, but until now there is no study to determine its protective roles in inflammatory bowel disease (IBD). The aim of this study was to reveal the in vitro gastrointestinal protective effects of BP against IBD using molecular and metabolic methods. Dextran sulfate sodium (DSS) challenged Caco-2 cell monolayers were applied to mimic intestinal epithelial cell dysfunctions and metabolic disorders. The pretreatment with BP extract rich in polyphenols ameliorated DSS-induced cell viability losses. It also exerted protective effects against intestinal barrier impairment by strengthening epithelial integrity and tight junction losses induced by DSS. BP up-regulated anti-oxidant (NQO1, Txnrd1, Nrf2) and down-regulated inflammatory (TNF-α and IL-6) mRNA expressions, in accompany with MAPK signaling inhibition. Furthermore, metabolomics analysis based on UPLC-Q-TOF/MS revealed that BP, and DSS treated Caco-2 cells have different metabolomic profiles, with significant changes on key metabolites involved in glycerophospholipid metabolism. Our results showed that BP has great therapeutic potential throughout the early stages of DSS-induced colitis.
Subject(s)
Bees/chemistry , Biological Products/pharmacology , Gastrointestinal Diseases/drug therapy , Intestines/drug effects , Pollen/chemistry , Protective Agents/pharmacology , Animals , Caco-2 Cells , Cell Line, Tumor , Dextran Sulfate/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gastrointestinal Diseases/chemically induced , Gastrointestinal Diseases/metabolism , Humans , Inflammation/chemically induced , Inflammation/drug therapy , Polyphenols/pharmacology , RNA, Messenger/metabolism , Signal Transduction/drug effects , Tight Junctions/drug effects , Up-Regulation/drug effectsABSTRACT
Honeys produced from medicinal plants hold great promise for human health. Herein, we determined the chemical composition and gastrointestinal protective effects of a novel monofloral honey from Prunella vulgaris (PVH). The physicochemical parameters (moisture, sugars, pH, protein content, diastase activity, and hydroxymethylfurfural) of the PVH samples met the criteria specified in European Union regulations and Chinese National Standards. Fifteen phenolic compounds were identified and quantified via high-performance liquid chromatography with a diode array detector and with time of flight tandem mass spectrometry detection (HPLC-DAD/Q-TOF-MS). Rosmarinic acid was found to be a potential marker for PVH identification. Using a dextran sulfate sodium (DSS)-induced acute colitis model, we demonstrated that the administration of PVH (5 g per kg b.w., p.o.) significantly decreased the disease activity index and mitigated colonic histopathological changes in rats. PVH also modulated the gut microbiota composition in the colitic rats, reversing the increase in the Bacteroidetes/Firmicutes ratio and restoring Lactobacillus spp. populations in DSS-challenged rats. The results of this study provide fundamental data on PVH, supporting its future application in the prevention of colitis.
Subject(s)
Colitis, Ulcerative/prevention & control , Dextran Sulfate/adverse effects , Gastrointestinal Microbiome/drug effects , Honey , Plants, Medicinal , Prunella/chemistry , Animals , Bacteroidetes , Colitis, Ulcerative/chemically induced , Colon/metabolism , Colon/pathology , Disease Models, Animal , Firmicutes , Flowers/chemistry , Lactobacillus , Male , Rats , Rats, Sprague-DawleyABSTRACT
BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.
Subject(s)
Antineoplastic Agents/pharmacology , BRCA2 Protein/deficiency , Drug Resistance, Neoplasm/physiology , E2F7 Transcription Factor/physiology , Enzyme Inhibitors/pharmacology , Neoplasm Proteins/physiology , CRISPR-Cas Systems , Cell Line, Tumor , DNA Replication/drug effects , DNA Replication/physiology , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , E2F7 Transcription Factor/deficiency , Gene Knockout Techniques , Genes, BRCA2 , Humans , Neoplasm Proteins/deficiency , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerases , Rad51 Recombinase/biosynthesis , Rad51 Recombinase/genetics , Recombinational DNA Repair/drug effects , Recombinational DNA Repair/physiologyABSTRACT
Bee pollen (BP) is collected by honeybees from flower pollen mixed with nectar and its secretions with extensive nutritional and therapeutic properties. Lipids are known to be critical contributors for the therapeutic effects of BP and vary depending on different plant sources; however, lipid profiles of BP are not available. Here, an UPLC-Q-Exactive Orbitrap/MS method was established for comprehensive lipidomics analysis of BP derived from three major nectar plants (Brassica campestris L., Camellia sinensis L., and Nelumbo nucifera Gaertn.). A total of nine lipid classes, including phosphatidylcholine (41 species), phosphatidylethanolamine (43 species), phosphatidylglycerol (9 species), phosphatidylserine (10 species), lysophosphatidylcholine (12 species), ceramide (8 species), diglyceride (27 species), triglyceride (137 species), and fatty acids (47 species), were first identified and quantified in the three BPs. In vitro anti-inflammatory activity was also discovered in the lipid extracts of three BPs, which has potential relevance to the abundance of phospholipids and unsaturated fatty acids in BP. Our comprehensive lipidomics profiling and in vitro anti-inflammatory properties of BP provide evidence for its future application.
Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Lipids/chemistry , Lipids/pharmacology , Pollen/chemistry , Animals , Bees , Chromatography, High Pressure Liquid , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Macrophages/drug effects , Macrophages/immunology , Mass Spectrometry , Mice , RAW 264.7 CellsABSTRACT
The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP(+)/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.
Subject(s)
Endoplasmic Reticulum Stress , Proline/biosynthesis , Saccharomyces cerevisiae/metabolism , NADP/metabolism , Phosphotransferases (Carboxyl Group Acceptor)/genetics , Phosphotransferases (Carboxyl Group Acceptor)/metabolism , Pyrroline Carboxylate Reductases/genetics , Pyrroline Carboxylate Reductases/metabolism , Saccharomyces cerevisiae/genetics , Unfolded Protein Response , delta-1-Pyrroline-5-Carboxylate ReductaseABSTRACT
SIGNIFICANCE: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. RECENT ADVANCES: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. CRITICAL ISSUES: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? FUTURE DIRECTIONS: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response.
Subject(s)
Oxidative Stress/physiology , Plants/metabolism , Proline/metabolism , Adaptation, Physiological , Metabolic Networks and PathwaysABSTRACT
BACKGROUND: Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative. RESULTS: We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity. CONCLUSION: Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation.
Subject(s)
Methionine Sulfoxide Reductases/chemistry , Methionine/analogs & derivatives , Methionine/chemistry , Alteromonadaceae/enzymology , Animals , Computational Biology , Cysteine/metabolism , Databases, Protein , Electrophoresis, Polyacrylamide Gel , Electrophoretic Mobility Shift Assay , Hydrogen Peroxide/chemistry , Methionine Sulfoxide Reductases/genetics , Methionine Sulfoxide Reductases/metabolism , Mice , Oxidation-Reduction , Pseudomonas putida/enzymology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Zea mays/enzymologyABSTRACT
Proline metabolism has an underlying role in apoptotic signaling that influences tumorigenesis. Proline is oxidized to glutamate in the mitochondria, with the rate-limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53, leading to increased proline oxidation, reactive oxygen species formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and maintained ATP and NADPH levels after exposure to hydrogen peroxide. Inhibition or siRNA-mediated knockdown of PRODH abolished proline protection against oxidative stress, whereas knockdown of Δ(1)-pyrroline-5-carboxylate reductase, a key enzyme in proline biosynthesis, had no impact on proline protection. Potential linkages between proline metabolism and signaling pathways were explored. The combined inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 eliminated proline protection. A significant increase in Akt activation was observed in proline-treated cells after hydrogen peroxide stress along with a corresponding increase in the phosphorylation of the forkhead transcription factor class O3a (FoxO3a). The role of PRODH in proline-mediated protection was validated in the prostate carcinoma cell line PC3. Knockdown of PRODH in PC3 cells attenuated phosphorylated levels of Akt and FoxO3a and decreased cell survival during hydrogen peroxide stress. The results provide evidence that PRODH is essential in proline protection against hydrogen peroxide-mediated cell death and that proline/PRODH helps activate Akt in cancer cells.