ABSTRACT
Maintaining genomic integrity and stability is crucial for life; yet, no tissue-driven mechanism that robustly safeguards the epithelial genome has been discovered. Epidermal stem cells (EpiSCs) continuously replenish the stratified layers of keratinocytes that protect organisms against various environmental stresses. To study the dynamics of DNA-damaged cells in tissues, we devised an in vivo fate tracing system for EpiSCs with DNA double-strand breaks (DSBs) and demonstrated that those cells exit from their niches. The clearance of EpiSCs with DSBs is caused by selective differentiation and delamination through the DNA damage response (DDR)-p53-Notch/p21 axis, with the downregulation of ITGB1. Moreover, concomitant enhancement of symmetric cell divisions of surrounding stem cells indicates that the selective elimination of cells with DSBs is coupled with the augmented clonal expansion of intact stem cells. These data collectively demonstrate that tissue autonomy through the dynamic coupling of cell-autonomous and non-cell-autonomous mechanisms coordinately maintains the genomic quality of the epidermis.
Subject(s)
Epidermis/metabolism , Genome , Stem Cells/cytology , Animals , Apoptosis/genetics , Cell Differentiation/genetics , Cell Division/genetics , Cell Proliferation/genetics , Clone Cells , DNA Breaks, Double-Stranded , DNA Damage/genetics , DNA Repair/genetics , Humans , Integrin beta1/metabolism , Mice, Inbred C57BL , Models, Biological , Receptors, Notch/metabolism , Signal Transduction/genetics , Stem Cell Niche , Stem Cells/metabolismABSTRACT
In selective autophagy, degradation targets are specifically recognized, sequestered by the autophagosome, and transported into the lysosome or vacuole. Previous studies delineated the molecular basis by which the autophagy machinery recognizes those targets, but the regulation of this process is still poorly understood. In this paper, we find that the highly conserved multifunctional kinase Hrr25 regulates two distinct selective autophagy-related pathways in Saccharomyces cerevisiae. Hrr25 is responsible for the phosphorylation of two receptor proteins: Atg19, which recognizes the assembly of vacuolar enzymes in the cytoplasm-to-vacuole targeting pathway, and Atg36, which recognizes superfluous peroxisomes in pexophagy. Hrr25-mediated phosphorylation enhances the interactions of these receptors with the common adaptor Atg11, which recruits the core autophagy-related proteins that mediate the formation of the autophagosomal membrane. Thus, this study introduces regulation of selective autophagy as a new role of Hrr25 and, together with other recent studies, reveals that different selective autophagy-related pathways are regulated by a uniform mechanism: phosphoregulation of the receptor-adaptor interaction.
Subject(s)
Autophagy/physiology , Casein Kinase I/metabolism , Receptors, Cell Surface/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/metabolism , Aminopeptidases/metabolism , Autophagy-Related Proteins , Binding Sites/genetics , COP-Coated Vesicles/metabolism , Casein Kinase I/genetics , GTPase-Activating Proteins/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Mutation , Peroxins , Peroxisomes/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Protein Transport , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.
Subject(s)
DNA-Binding Proteins/metabolism , Kinesins/metabolism , Mitosis , Xeroderma Pigmentosum/genetics , Xeroderma Pigmentosum/metabolism , Animals , Cell Nucleus/metabolism , DNA Repair , DNA-Binding Proteins/genetics , Gene Knockout Techniques , HEK293 Cells , Humans , Kinesins/genetics , MiceABSTRACT
Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH. Instead, it included FAM96B (now designated MIP18), Ciao1, and ANT2. MMS19, MIP18, and XPD localized to the mitotic spindle during mitosis. The siRNA-mediated knockdown of MMS19, MIP18, or XPD led to improper chromosome segregation and the accumulation of nuclei with abnormal shapes. In addition, the frequency of abnormal mitosis and nuclei was increased in XP-D and XP-D/CS patients' cells. These results indicate that the MMS19-XPD protein complex, now designated MMXD (MMS19-MIP18-XPD), is required for proper chromosome segregation, an abnormality of which could contribute to the pathogenesis in some cases of XP-D and XP-D/CS.
Subject(s)
Carrier Proteins/metabolism , Chromosome Segregation , Nuclear Proteins/metabolism , Transcription Factor TFIIH/metabolism , Transcription Factors/metabolism , Xeroderma Pigmentosum Group D Protein/metabolism , Xeroderma Pigmentosum/genetics , Adenine Nucleotide Translocator 2/metabolism , Binding Sites , Carrier Proteins/genetics , Cell Nucleus Shape , Gene Knockdown Techniques , HCT116 Cells , HeLa Cells , Humans , Metallochaperones/metabolism , Metalloproteins , Microscopy, Fluorescence , Mitosis , Multiprotein Complexes , Nuclear Proteins/genetics , Protein Interaction Domains and Motifs , Protein Interaction Mapping , RNA Interference , Spindle Apparatus/metabolism , Transcription Factors/genetics , Transfection , Xeroderma Pigmentosum/metabolism , Xeroderma Pigmentosum/pathology , Xeroderma Pigmentosum Group D Protein/geneticsABSTRACT
Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori.
