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1.
Transl Res ; 237: 1-15, 2021 11.
Article in English | MEDLINE | ID: mdl-34004371

ABSTRACT

The 7 members of the A3 family of cytidine deaminases (A3A to A3H) share a conserved catalytic activity that converts cytidines in single-stranded (ss) DNA into uridines, thereby inducing mutations. After their initial identification as cell-intrinsic defenses against HIV and other retroviruses, A3s were also found to impair many additional viruses. Moreover, some of the A3 proteins (A3A, A3B, and A3H haplotype I) are dysregulated in cancer cells, thereby causing chromosomal mutations that can be selected to fuel progression of malignancy. Viral mechanisms that increase transcription of A3 genes or induce proteasomal degradation of A3 proteins have been characterized. However, only a few underlying biological mechanisms regulating levels of A3s in uninfected cells have been described. Here, we characterize that the von Hippel-Lindau tumor suppressor (pVHL), via its CRLpVHL, induces degradation of all 7 A3 proteins. Two independent lines of evidence supported the conclusion that the multiprotein CRLpVHL complex is necessary for A3 degradation. CRLpVHL more effectively induced degradation of nuclear, procancer A3 (A3B) than the cytoplasmic, antiretroviral A3 (A3G). These results identify specific cellular factors that regulate A3s post-translationally.


Subject(s)
APOBEC Deaminases/metabolism , Gene Expression Regulation, Enzymologic/physiology , Ubiquitin-Protein Ligases/metabolism , APOBEC Deaminases/genetics , Cell Line, Tumor , Down-Regulation , Humans , Ubiquitin-Protein Ligases/genetics
2.
Autophagy ; 15(3): 527-542, 2019 03.
Article in English | MEDLINE | ID: mdl-30335568

ABSTRACT

50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs. Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type.


Subject(s)
Autophagy/genetics , Escherichia coli Infections/metabolism , Urinary Tract Infections/metabolism , Uropathogenic Escherichia coli , Urothelium/microbiology , Animals , Autophagy-Related Protein 7/genetics , Autophagy-Related Protein 7/metabolism , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Cell Line , Epithelial Cells/metabolism , Escherichia coli Infections/genetics , Escherichia coli Infections/microbiology , Escherichia coli Infections/pathology , Female , Genetic Variation , Humans , Macrophages/metabolism , Mice , Mice, Knockout , Multivesicular Bodies/genetics , Multivesicular Bodies/microbiology , Multivesicular Bodies/pathology , Urinary Bladder/microbiology , Urinary Tract Infections/genetics , Urinary Tract Infections/microbiology , Urinary Tract Infections/pathology , Uroplakins/metabolism , Urothelium/cytology , Urothelium/metabolism , Urothelium/ultrastructure , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism
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