Revealing novel telomere proteins using in vivo cross-linking, tandem affinity purification, and label-free quantitative LC-FTICR-MS.

Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be identified by this approach. The success of this approach will allow us to create a more complete understanding of telomere maintenance and have broad applicability.

SCKs as nanoparticle carriers of doxorubicin: investigation of core composition on the loading, release and cytotoxicity profiles.

SCK nanoparticles having differing core thermal characteristics were designed and evaluated as thermoresponsive drug delivery systems of doxorubicin for the killing of cancerous cells.

Alternative lengthening of telomeres (ALT) and chromatin: is there a connection?

The acquisition of cellular immortality is a critical step in the tumorigenic process that requires stabilization of the telomeres, nucleoprotein structures at the termini of chromosomes. While the majority of human tumors stabilize their telomeres through activation of telomerase (hTERT), a significant portion (10-15%) utilize a poorly understood alternative mechanism of telomere maintenance referred to as ALT (Alternative Lengthening of Telomeres). Strikingly, the ALT mechanism is more prevalent in tumors arising from tissues of mesenchymal origin than in those of epithelial origin. This observation suggests that cell type specific mechanisms favor the activation of the ALT mechanism versus telomerase in human tumorigenesis. In addition, the presence of an alternative mechanism of telomere maintenance raises the possibility that telomerase-positive tumors undergoing anti-telomerase therapies might escape by activating the ALT pathway. For these reasons, delineating the ALT mechanism is critical for our understanding of the tumorigenic process and the development of ALT-specific anti-neoplastic therapies. Recent studies have demonstrated that epigenetic modifications at telomeres have a profound effect on telomere length, and may also be linked to the ALT mechanism. In this review we focus on these recent advances and their implications in telomere maintenance.

Role of copper in the proteosome-mediated degradation of the multicopper oxidase hephaestin.

To elucidate the mechanisms of cuproprotein biosynthesis in the secretory pathway, a polyclonal antiserum was generated against hephaestin, a multicopper oxidase essential for enteric iron absorption. Immunoblot analysis and pulse-chase metabolic labeling revealed that hephaestin is synthesized as a single-chain polypeptide modified by N-linked glycosylation to a mature 161-kDa species. Cell surface biotinylation and immunofluorescent studies of polarized, differentiated colon carcinoma cells detected hephaestin on the basolateral surface under steady-state conditions. However, a decrease in the intracellular copper concentration resulted in a marked diminution in the abundance of this protein. Metabolic studies revealed no effect of decreased intracellular copper on the rate of hephaestin synthesis but a dramatic, specific, and reproducible increase in the turnover of the mature 161-kDa protein. Surprisingly, inhibitor studies revealed that this turnover occurs exclusively in the proteasome, and consistent with this finding, in vitro studies identified polyubiquitinated hephaestin under conditions abrogating copper incorporation into this protein. Taken together, these studies demonstrate the presence of a quality control system for posttranslational protein modification occurring beyond the endoplasmic reticulum that, in the case of hephaestin, directly links the rate of enteric iron uptake to nutritional copper status.

The copper-iron connection: hereditary aceruloplasminemia.

Hereditary aceruloplasminemia is an autosomal recessive disorder of iron homeostasis due to loss-of-function mutations in the ceruloplasmin gene. Affected individuals may present in adulthood with evidence of hepatic iron overload, diabetes, peripheral retinal degeneration, dystonia, dementia, or dysarthria. Laboratory studies demonstrate microcytic anemia, elevated serum ferritin, and a complete absence of serum ceruloplasmin ferroxidase activity. Consistent with the observed neurologic findings, magnetic resonance imaging reveals iron accumulation within the basal ganglia. Histologic studies detect abundant iron in hepatocytes, reticuloendothelial cells of the liver and spleen, beta cells of the pancreas, and astrocytes and neurons throughout the central nervous system. Characterization of this disorder reveals an essential role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores and provides new insights into the mechanisms of human iron metabolism.