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Query: UNIPROT:P06889 (Mol)
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Proteins are frequently processed by proteases in cell signaling pathways to perform their biological functions in response to environmental stimuli. Identification of the exact cleavage sites provides necessary information for the study of their biological functions. Although proteomic approaches for profiling of protein N-termini have been developed extensively in the past few years, the N-terminal profiling strategy has its inherent disadvantages. Therefore, C-terminal profiling approaches might be a complementary approach for the identification of protein cleavages although it has similar shortcomings as N-terminal profiling methods. In this protocol, we describe an approach, termed ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling, for affinity labeling of protein C-termini for a protein or proteome. This method uses the transpeptidase activity of carboxypeptidase Y to label protein C-termini with an affinity biotin tag for subsequent isolation with avidin beads and identification by mass spectrometer. It is complementary to the N-terminal profiling approaches and can be used to identify proteolytic cleavages for a specific protease or in cell signaling events, such as apoptosis.
Methods Mol Biol 2017
PMID:ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling. 2831 48

Chronic oxidative stress is the major endogenous metabolic stress and contributes directly to telomere shortening and senescence. Understanding the dysfunction of telomeres under oxidative stress will greatly facilitate healthy aging and advance the treatment of aging-related diseases. Here, we describe the KR-TEL (KillerRed induced DNA damage at telomeres) system that induces site-specific oxidative damage at telomeres. We have developed the KR-TEL system by fusing killerred with the shelterin component TRF1 (KR-TRF1) or other shelterin proteins. Killerred (KR), an engineered red fluorescent chromophore, is capable of generating site-specific superoxide upon green light activation (550-580 nm). When KR-TRF1 expressing cells are exposed to green or laser light at defined wavelength to activate KR, localized oxidative DNA damage will be induced at telomeres. KR-induced oxidative DNA damage shows a high degree of resemblance to the complex spectrum of DNA damage induced by radiation in terms of the ratios of oxidized bases and DNA strand breaks. Unlike current oxidation-inducing methods (e.g., IR, chemical, and toxicants), which create damage throughout the genome, KR produces spatially limited oxidative DNA damage only in its immediate proximity. This property of KR allows us to engineer oxidative damage specifically at the telomere in a light dose-dependent manner. Using the KR-TEL system to determine the DNA damage response and repair mechanisms at telomeres has several advantages, which make it an ideal system to investigate the mechanism of how telomere integrity is maintained and how this mechanism plays a role in cancer biology.
Methods Mol Biol 2017
PMID:Induction of Site-Specific Oxidative Damage at Telomeres by Killerred-Fused Shelretin Proteins. 2832 6

TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.
Mol Cell Biol 2019 11 01
PMID:TIN2 Functions with TPP1/POT1 To Stimulate Telomerase Processivity. 3138 50

Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1S) and the amino acid L104 outside the TEL patch, TPP1's telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1S (TPP1S knockout [KO]), as well as the complete TPP1 KO cell lines, undergo telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1L104A/L104A mutant cells, which have short and stable telomeres similar to the TPP1S KO. In contrast to TPP1S KO cells, TPP1L104A/L104A cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1L104A/L104A shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1L104A/L104A as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point.
Mol Biol Cell 2020 11 01
PMID:Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1. 3290 38


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