The orderly recruitment, retention, and disassembly of DNA damage response proteins at sites of damaged DNA is a conserved process throughout eukaryotic evolution. The recruitment and retention of DNA repair factors in foci is mediated by a complex network of protein-protein interactions; however, the mechanisms of focus disassembly remain to be defined. Mediator of DNA damage checkpoint protein 1 (MDC1) is an early and key component of the genome surveillance network activated by DNA double-strand breaks (DSBs). Here, we investigated the disassembly of MDC1 foci. First, we show that ubiquitylation directs the MDC1 protein for proteasome-dependent degradation. Ubiquitylated MDC1 associates with chromatin before and after exposure of cells to ionizing radiation (IR). In addition, increased MDC1 ubiquitylation in the chromatin fraction is observed in response to IR, which is correlated with a reduction in total MDC1 protein levels. We demonstrate that blocking MDC1 degradation by proteasome inhibitors leads to a persistence of MDC1 foci. Consistent with this observation, chromatin immunoprecipitation experiments reveal increased MDC1 protein at site-specific DSBs. Interestingly, we show that the persistence of MDC1 foci is associated with an abrogated recruitment of the downstream factor BRCA1 in a manner that is RNF8 independent. Collectively, the evidence presented here supports a novel mechanism for the disassembly of MDC1 foci via ubiquitin-proteasome dependent degradation, which appears to be a key step for the efficient assembly of BRCA1 foci.

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO ؊ ) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO ؊ andHOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.

Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin ␣ v ␤ 3 and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO ؊ ) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO ؊ treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO ؊ treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO ؊ prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.Elastic fibers are key extracellular matrix structures that provide the stretch and recoil properties of tissues such as arteries, lungs, and skin. Elastic fibers consist of two major components, elastin and microfibrils. Elastin is the predominant component of elastic fibers, comprising Ͼ90% of the total mass. Tropoelastin (TE), 2 the soluble precursor of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, endothelial cells, and smooth muscle cells (SMCs). Assembly of monomeric TE into elastic fibers is a multistep process. Upon secretion from cells, TE monomers organize into aggregates on the cell surface. These aggregates are then deposited onto pre-existing microfibrils. Microfibrillar components align the TE aggregates, which undergo cross-linking to form mature elastic fibers. Cell surface molecules, such as the 67-kDa elastin-binding protein (EBP), ␣ v ␤ 3 integrin, and glycosaminoglycans (GAGs), have been proposed to be involved in elastic fiber assembly either by promoting aggregation of TE monomers or by keeping the microfibrils close to the cell membrane through interactions with microfibrillar components (1-5).TE is mainly synthesized during late fetal and early postnatal stages of development. Synthesis of TE in normal adult tissues is negligible; however, in several cardiovascular and pulmonary diseases, such as atherosclerosis and emphysema/chronic obstr...

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Abstract. Cancer stem cells (CSCs) are rare tumor cells that have the potential to proliferate, self-renew and induce tumorigenesis. Over the past few years, CSCs have been isolated from several different tumors and when implanted into immune-deficient mice, generate tumors that are identical to the parental tumors. In this review, we summarize the current literature on CSCs, which suggests that since these cells have the ability to drive tumor formation, specifically targeting them may lead to more effective therapies against tumors.