5 research outputs found
Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes
ATM is a protein kinase that initiates a well-characterized
signaling cascade in cells exposed to ionizing radiation (IR). However,
the role for ATM in coordinating critical protein interactions and
subsequent exchanges within DNA damage response (DDR) complexes is
unknown. We combined SILAC-based tandem mass spectrometry and a subcellular
fractionation protocol to interrogate the proteome of irradiated cells
treated with or without the ATM kinase inhibitor KU55933. We developed
an integrative network analysis to identify and prioritize proteins
that were responsive to KU55933, specifically in chromatin, and that
were also enriched for physical interactions with known DNA repair
proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as
strong candidates. Using fluorescence recovery after photobleaching,
we found that the exchange of GFP-53BP1 in DDR complexes decreased
with KU55933. Further, we found that ANXA1 knockdown sensitized cells
to IR via a mechanism that was not potentiated by KU55933. Our study
reveals a role for ATM kinase activity in the dynamic exchange of
proteins in DDR complexes and identifies a role for ANXA1 in cellular
radioprotection
Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes
ATM is a protein kinase that initiates a well-characterized
signaling cascade in cells exposed to ionizing radiation (IR). However,
the role for ATM in coordinating critical protein interactions and
subsequent exchanges within DNA damage response (DDR) complexes is
unknown. We combined SILAC-based tandem mass spectrometry and a subcellular
fractionation protocol to interrogate the proteome of irradiated cells
treated with or without the ATM kinase inhibitor KU55933. We developed
an integrative network analysis to identify and prioritize proteins
that were responsive to KU55933, specifically in chromatin, and that
were also enriched for physical interactions with known DNA repair
proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as
strong candidates. Using fluorescence recovery after photobleaching,
we found that the exchange of GFP-53BP1 in DDR complexes decreased
with KU55933. Further, we found that ANXA1 knockdown sensitized cells
to IR via a mechanism that was not potentiated by KU55933. Our study
reveals a role for ATM kinase activity in the dynamic exchange of
proteins in DDR complexes and identifies a role for ANXA1 in cellular
radioprotection
Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes
ATM is a protein kinase that initiates a well-characterized
signaling cascade in cells exposed to ionizing radiation (IR). However,
the role for ATM in coordinating critical protein interactions and
subsequent exchanges within DNA damage response (DDR) complexes is
unknown. We combined SILAC-based tandem mass spectrometry and a subcellular
fractionation protocol to interrogate the proteome of irradiated cells
treated with or without the ATM kinase inhibitor KU55933. We developed
an integrative network analysis to identify and prioritize proteins
that were responsive to KU55933, specifically in chromatin, and that
were also enriched for physical interactions with known DNA repair
proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as
strong candidates. Using fluorescence recovery after photobleaching,
we found that the exchange of GFP-53BP1 in DDR complexes decreased
with KU55933. Further, we found that ANXA1 knockdown sensitized cells
to IR via a mechanism that was not potentiated by KU55933. Our study
reveals a role for ATM kinase activity in the dynamic exchange of
proteins in DDR complexes and identifies a role for ANXA1 in cellular
radioprotection
Table_1_Attenuated IL-2 muteins leverage the TCR signal to enhance regulatory T cell homeostasis and response in vivo.xlsx
Interleukin-2 (IL-2), along with T-cell receptor (TCR) signaling, are required to control regulatory T cell (Treg) homeostasis and function in vivo. Due to the heightened sensitivity to IL-2, Tregs retain the ability to respond to low-dose or attenuated forms of IL-2, as currently being developed for clinical use to treat inflammatory diseases. While attenuated IL-2 increases Treg selectivity, the question remains as to whether a weakened IL-2 signal sufficiently enhances Treg suppressive function(s) toward disease modification. To understand this question, we characterized the in vivo activity and transcriptomic profiles of two different attenuated IL-2 muteins in comparison with wildtype (WT) IL-2. Our study showed that, in addition to favoring Tregs, the attenuated muteins induced disproportionately robust effects on Treg activation and conversion to effector Treg (eTreg) phenotype. Our data furthermore suggested that Tregs activated by attenuated IL-2 muteins showed reduced dependence on TCR signal, at least in part due to the enhanced ability of IL-2 muteins to amplify the TCR signal in vivo. These results point to a new paradigm wherein IL-2 influences Tregs’ sensitivity to antigenic signal, and that the combination effect may be leveraged for therapeutic use of attenuated IL-2 muteins.</p
DataSheet_1_Attenuated IL-2 muteins leverage the TCR signal to enhance regulatory T cell homeostasis and response in vivo.pdf
Interleukin-2 (IL-2), along with T-cell receptor (TCR) signaling, are required to control regulatory T cell (Treg) homeostasis and function in vivo. Due to the heightened sensitivity to IL-2, Tregs retain the ability to respond to low-dose or attenuated forms of IL-2, as currently being developed for clinical use to treat inflammatory diseases. While attenuated IL-2 increases Treg selectivity, the question remains as to whether a weakened IL-2 signal sufficiently enhances Treg suppressive function(s) toward disease modification. To understand this question, we characterized the in vivo activity and transcriptomic profiles of two different attenuated IL-2 muteins in comparison with wildtype (WT) IL-2. Our study showed that, in addition to favoring Tregs, the attenuated muteins induced disproportionately robust effects on Treg activation and conversion to effector Treg (eTreg) phenotype. Our data furthermore suggested that Tregs activated by attenuated IL-2 muteins showed reduced dependence on TCR signal, at least in part due to the enhanced ability of IL-2 muteins to amplify the TCR signal in vivo. These results point to a new paradigm wherein IL-2 influences Tregs’ sensitivity to antigenic signal, and that the combination effect may be leveraged for therapeutic use of attenuated IL-2 muteins.</p