Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG) that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity of E. coli TDG homolog (EcMUG), which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2

AbstractThe yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18Ubc9, the Xenopus homolog of Ubc9p, associates specifically with p88RanGAP1 and p340RanBP2[2]. Ran-binding protein 2 (p340RanBP2) is a nuclear pore protein [3,4], and p88RanGAP1 is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5–7], and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 [2,5,6]. Here we show that p18Ubc9 acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18Ubc9 interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts

SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER

Cell-derived Giant Membrane Vesicles Enclosing the Anthracycline Anti-Cancer Drug Doxorubicin and their Cytotoxicity

Abstract: Although giant membrane vesicles prepared from paraformaldehyde-treated mammalian cells have been used to elucidate lipid and protein dynamics on the cell surface, there are few studies that have used them as a tool to deliver drugs to cells. Here we found that anti-cancer drug doxorubicin (Dox) was efficiently incorporated into and stably retained in giant membrane vesicles prepared from HeLa human cervical cancer cells. Intriguingly, coincubation of Dox-enclosed giant membrane vesicles with human pancreatic cancer-derived PK-59 and gastric cancer-derived KE-39 cells led to cell death. Microscopic observation of vesicle-docked cells revealed that docking of at least one to a few Dox-enclosed giant vesicles on the cellular cortex was sufficient to induce cell death, suggesting applicability of cell-derived giant membrane vesicles as an efficient drug delivery vector in cultured cell systems. Keywords: Anti-cancer treatment, Extracellular vesicle, Doxorubicin, Drug delivery

Enzymes of the SUMO Modification Pathway Localize to Filaments of the Nuclear Pore Complex

SUMOs are small ubiquitin-related polypeptides that are reversibly conjugated to many nuclear proteins. Although the number of identified substrates has grown rapidly, relatively little is still understood about when, where, and why most proteins are modified by SUMO. Here, we demonstrate that enzymes involved in the SUMO modification and demodification of proteins are components of the nuclear pore complex (NPC). We show that SENP2, a SUMO protease that is able to demodify both SUMO-1 and SUMO-2 or SUMO-3 protein conjugates, localizes to the nucleoplasmic face of the NPC. The unique amino-terminal domain of SENP2 interacts with the FG repeat domain of Nup153, indicating that SENP2 associates with the nucleoplasmic basket of the NPC. We also investigated the localization of the SUMO conjugating enzyme, Ubc9. Using immunogold labeling of isolated nuclear envelopes, we found that Ubc9 localizes to both the cytoplasmic and the nucleoplasmic filaments of the NPC. In vitro binding studies revealed that Ubc9 and SUMO-1-modified RanGAP1 bind synergistically to form a trimeric complex with a component of the cytoplasmic filaments of the NPC, Nup358. Our results indicate that both SUMO modification and demodification of proteins may occur at the NPC and suggest a connection between the SUMO modification pathway and nucleocytoplasmic transport

The Transcriptional Cofactor MCAF1/ATF7IP Is Involved in Histone Gene Expression and Cellular Senescence

<div><p>Cellular senescence is post-mitotic or oncogene-induced events combined with nuclear remodeling. MCAF1 (also known as hAM or ATF7IP), a transcriptional cofactor that is overexpressed in various cancers, functions in gene activation or repression, depending on interacting partners. In this study, we found that MCAF1 localizes to PML nuclear bodies in human fibroblasts and non-cancerous cells. Interestingly, depletion of MCAF1 in fibroblasts induced premature senescence that was characterized by cell cycle arrest, SA-β-gal activity, and senescence-associated heterochromatic foci (SAHF) formation. Under this condition, core histones and the linker histone H1 significantly decreased at both mRNA and protein levels, resulting in reduced nucleosome formation. Consistently, in activated Ras-induced senescent fibroblasts, the accumulation of MCAF1 in PML bodies was enhanced via the binding of this protein to SUMO molecules, suggesting that sequestration of MCAF1 to PML bodies promotes cellular senescence. Collectively, these results reveal that MCAF1 is an essential regulator of cellular senescence.</p> </div

MCAF1 knockdown induces cell cycle arrest.

<p>(<b>A</b>) IMR90 cells were treated with the indicated siRNAs for 48 hr and analyzed by Western blotting with anti-MCAF1 and anti-β-tubulin antibodies. The images were quantitatively assessed by densitometry. (<b>B</b>) Growth curves for siControl, siMCAF1-1, and siMCAF1-2 cells. (<b>C</b>) EdU incorporation assay in control and MCAF1 knockdown cells. **P<0.01. (D) RT-qPCR analysis of the cell cycle genes at 48 hr after siRNA treatment. **P<0.01.</p

Knockdown of MCAF1 induces premature senescence.

<p>(<b>A</b>) Western blot analysis of the cdk inhibitors p16 and p21 and RB proteins in control and MCAF1 knockdown IMR90 cells. The images were quantitatively assessed by densitometry. (<b>B</b>, <b>C</b>) IMR90 cells were treated with the indicated siRNAs, and analyzed for SA-β-gal activity (B) or the formation of SAHF (C) at 8 days after siRNA treatment. **P<0.01. (<b>D</b>) Immunofluorescence analysis of MCAF1 and MacroH2A in control and SAHF-positive MCAF1 knockdown cells. DNA was stained with DAPI. Scale bar: 10 µM.</p