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1.
Dev Cell ; 59(6): 759-775.e5, 2024 Mar 25.
Article in English | MEDLINE | ID: mdl-38354739

ABSTRACT

Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that directly binds vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy, resulting in impaired caloric restriction-induced longevity. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we identify the tethering machinery of vCLIP and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy.


Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolism , Lipid Droplets/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vacuoles/metabolism , Lipid Metabolism/physiology , Autophagy
2.
Science ; 376(6600): 1471-1476, 2022 06 24.
Article in English | MEDLINE | ID: mdl-35737787

ABSTRACT

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.


Subject(s)
DNA Damage , DNA Glycosylases , DNA Repair , Oxidative Stress , Biocatalysis/drug effects , DNA Damage/drug effects , DNA Glycosylases/chemistry , DNA Glycosylases/drug effects , DNA Repair/drug effects , Enzyme Activation , Glycine/chemistry , Humans , Ligands , Oxidative Stress/genetics , Phenylalanine/chemistry , Substrate Specificity
3.
PLoS Genet ; 17(11): e1009911, 2021 11.
Article in English | MEDLINE | ID: mdl-34780474

ABSTRACT

The capacity of a cell to maintain proteostasis progressively declines during aging. Virtually all age-associated neurodegenerative disorders associated with aggregation of neurotoxic proteins are linked to defects in the cellular proteostasis network, including insufficient lysosomal hydrolysis. Here, we report that proteotoxicity in yeast and Drosophila models for Parkinson's disease can be prevented by increasing the bioavailability of Ca2+, which adjusts intracellular Ca2+ handling and boosts lysosomal proteolysis. Heterologous expression of human α-synuclein (αSyn), a protein critically linked to Parkinson's disease, selectively increases total cellular Ca2+ content, while the levels of manganese and iron remain unchanged. Disrupted Ca2+ homeostasis results in inhibition of the lysosomal protease cathepsin D and triggers premature cellular and organismal death. External administration of Ca2+ reduces αSyn oligomerization, stimulates cathepsin D activity and in consequence restores survival, which critically depends on the Ca2+/calmodulin-dependent phosphatase calcineurin. In flies, increasing the availability of Ca2+ discloses a neuroprotective role of αSyn upon manganese overload. In sum, we establish a molecular interplay between cathepsin D and calcineurin that can be activated by Ca2+ administration to counteract αSyn proteotoxicity.


Subject(s)
Calcineurin/genetics , Cathepsin D/genetics , Parkinson Disease/genetics , alpha-Synuclein/genetics , Aging/drug effects , Aging/genetics , Animals , Animals, Genetically Modified/genetics , Calcium/metabolism , Calcium/pharmacology , Cell Death/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation/drug effects , Humans , Lysosomes/drug effects , Lysosomes/genetics , Neurons/drug effects , Parkinson Disease/metabolism , Parkinson Disease/pathology , Protein Aggregation, Pathological/drug therapy , Protein Aggregation, Pathological/genetics , Protein Aggregation, Pathological/pathology , Proteolysis/drug effects , Saccharomyces cerevisiae/genetics
4.
Nucleic Acids Res ; 47(10): 5276-5292, 2019 06 04.
Article in English | MEDLINE | ID: mdl-30976810

ABSTRACT

Abasic (AP) sites, the most common DNA lesions are frequently associated with double strand breaks (DSBs) and can pose a block to the final ligation. In many prokaryotes, nonhomologous end joining (NHEJ) repair of DSBs relies on a two-component machinery constituted by the ring-shaped DNA-binding Ku that recruits the multicatalytic protein Ligase D (LigD) to the ends. By using its polymerization and ligase activities, LigD fills the gaps that arise after realignment of the ends and seals the resulting nicks. Here, we show the presence of a robust AP lyase activity in the polymerization domain of Bacillus subtilis LigD (BsuLigD) that cleaves AP sites preferentially when they are proximal to recessive 5'-ends. Such a reaction depends on both, metal ions and the formation of a Watson-Crick base pair between the incoming nucleotide and the templating one opposite the AP site. Only after processing the AP site, and in the presence of the Ku protein, BsuLigD catalyzes both, the in-trans addition of the nucleotide to the 3'-end of an incoming primer and the ligation of both ends. These results imply that formation of a preternary-precatalytic complex ensures the coupling of AP sites cleavage to the end-joining reaction by the bacterial LigD.


Subject(s)
Bacillus subtilis/enzymology , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA Ligase ATP/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Binding Sites , Catalysis , Catalytic Domain , DNA, Bacterial/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli/enzymology , Humans , Ions , Ku Autoantigen/metabolism , Mutagenesis, Site-Directed , Nucleotides/metabolism , Oligonucleotides , Protein Binding , Protein Domains
5.
Sci Rep ; 7(1): 6907, 2017 07 31.
Article in English | MEDLINE | ID: mdl-28761124

ABSTRACT

Phaeocystis globosa virus 16T is a giant virus that belongs to the so-called nucleo-cytoplasmic large DNA virus (NCLDV) group. Its linear dsDNA genome contains an almost full complement of genes required to participate in viral base excision repair (BER). Among them is a gene coding for a bimodular protein consisting of an N-terminal Polß-like core fused to a C-terminal domain (PgVPolX), which shows homology with NAD+-dependent DNA ligases. Analysis of the biochemical features of the purified enzyme revealed that PgVPolX is a multifunctional protein that could act as a "Swiss army knife" enzyme during BER since it is endowed with: 1) a template-directed DNA polymerization activity, preferentially acting on DNA structures containing gaps; 2) 5'-deoxyribose-5-phosphate (dRP) and abasic (AP) site lyase activities; and 3) an NAD+-dependent DNA ligase activity. We show how the three activities act in concert to efficiently repair BER intermediates, leading us to suggest that PgVPolX may constitute, together with the viral AP-endonuclease, a BER pathway. This is the first time that this type of protein fusion has been demonstrated to be functional.


Subject(s)
DNA Repair , DNA Viruses/enzymology , DNA-Directed DNA Polymerase/metabolism , DNA Ligases/chemistry , DNA Ligases/metabolism , DNA Replication , DNA Viruses/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Genome, Viral , Viral Proteins/chemistry , Viral Proteins/metabolism
6.
Nucleic Acids Res ; 44(4): 1833-44, 2016 Feb 29.
Article in English | MEDLINE | ID: mdl-26826709

ABSTRACT

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5'-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2'-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs.


Subject(s)
Bacillus subtilis/enzymology , DNA End-Joining Repair/genetics , DNA Ligases/genetics , Phosphorus-Oxygen Lyases/genetics , DNA Breaks, Double-Stranded , DNA Ligases/metabolism , DNA Repair/genetics , Phosphorus-Oxygen Lyases/metabolism , Pseudomonas aeruginosa/enzymology
7.
Nucleic Acids Res ; 42(21): 13082-95, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25355514

ABSTRACT

Intracellular reactive oxygen species as well as the exposure to harsh environmental conditions can cause, in the single chromosome of Bacillus subtilis spores, the formation of apurinic/apyrimidinic (AP) sites and strand breaks whose repair during outgrowth is crucial to guarantee cell viability. Whereas double-stranded breaks are mended by the nonhomologous end joining (NHEJ) system composed of an ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku, repair of AP sites would rely on an AP endonuclease or an AP-lyase, a polymerase and a ligase. Here we show that B. subtilis Ku (BsuKu), along with its pivotal role in allowing joining of two broken ends by B. subtilis LigD (BsuLigD), is endowed with an AP/deoxyribose 5'-phosphate (5'-dRP)-lyase activity that can act on ssDNA, nicked molecules and DNA molecules without ends, suggesting a potential role in BER during spore outgrowth. Coordination with BsuLigD makes possible the efficient joining of DNA ends with near terminal abasic sites. The role of this new enzymatic activity of Ku and its potential importance in the NHEJ pathway is discussed. The presence of an AP-lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa allows us to expand our results to other bacterial Ku proteins.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , DNA End-Joining Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-Binding Proteins/metabolism , Phosphorus-Oxygen Lyases/metabolism , DNA/metabolism , DNA Ligases/metabolism
8.
Int J Food Microbiol ; 165(3): 251-8, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23800737

ABSTRACT

Fusarium verticillioides and Fusarium proliferatum are important phytopathogens which contaminate cereals in the Mediterranean climatic region with fumonisins. In this study we examined the interaction between the fungicide efficacy of tebuconazole and water potential (Ψw) (-0.7-7.0MPa)×temperature (20-35°C) on growth and FUM1 gene expression by real time RT-PCR (an indicator of fumonisin biosynthesis) in strains of both Fusarium species. Concentrations of tebuconazole required to reduce growth by 50 and 90% (ED50 and ED90 values) were determined. Growth of strains of both species was largely reduced by tebuconazole, with similar efficacy profiles in the interacting water potential×temperature conditions. In contrast, FUM1 expression was not generally reduced by tebuconazole. Moreover, sub-lethal doses in combination with mild water stress and temperatures less than 35°C significantly induced FUM1 expression with slight differences in both species. These results suggest that the efficacy of antifungal compounds to reduce mycotoxin risk would be more effective if consideration is given to both growth rate and toxin biosynthesis in relation to interacting environmental conditions. This is the first study linking fungicide efficacy of tebuconazole with environmental factor effects on control of growth and FUM1 gene expression of F. verticillioides and F. proliferatum.


Subject(s)
Antifungal Agents/pharmacology , Environment , Fumonisins/metabolism , Fusarium/drug effects , Gene Expression Regulation, Fungal , Triazoles/pharmacology , Analysis of Variance , Fungicides, Industrial/pharmacology , Fusarium/genetics , Fusarium/growth & development , Fusarium/metabolism , Inhibitory Concentration 50 , Reverse Transcriptase Polymerase Chain Reaction , Temperature , Water/pharmacology
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