Summary The histone deacetylase SIRT6 promotes DNA repair, but its activity declines with age, with a concomitant accumulation of DNA damage. Furthermore, SIRT6 knockout mice exhibit an accelerated aging phenotype and die prematurely. Here, we report that brain-specific SIRT6-deficient mice survive, but present behavioral defects with major learning impairments by 4 months of age. Moreover, the brains of these mice show increased signs of DNA damage, cell death and hyperphosphorylated Tau – a critical mark in several neurodegenerative diseases. Mechanistically, SIRT6 regulates Tau protein stability and phosphorylation through increased activation of the kinase GSK3α/β. Finally, SIRT6 mRNA and protein levels are reduced in patients with Alzheimer’s disease. Taken together, our results suggest that SIRT6 is critical to maintain genomic stability in the brain and its loss leads to toxic Tau stability and phosphorylation. Therefore, SIRT6 and its downstream signaling could be targeted in Alzheimer’s disease and age related neurodegeneration.
DNA double-strand breaks (DSB) are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure that has high affinity for DSB. SIRT6 relocates to sites of damage independently of signaling and known sensors. It activates downstream signaling for DSB repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the homologous recombination and non-homologous end joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as a DNA damage sensor, a critical factor in initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB-binding capacity and DDR activation. SIRT6 activates the DDR before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 as a sensor of DSB, and pave the road to dissecting the contributions of distinct DSB sensors in downstream signaling.
Background-Chemokine-chemokine receptor interaction and the subsequent recruitment of T-lymphocytes to the graft are early events in the development of chronic rejection of transplanted hearts or cardiac allograft vasculopathy (CAV).In this study, we sought to determine whether blockade of chemokine receptors CCR1 and CCR5 with Met-RANTES affects the development of CAV in a murine model. Methods and Results-B6.CH-2 bm12 strain donor hearts were transplanted heterotopically into wild-type C57BL/6 mice (myosin heavy chain II mismatch). Recipients were treated daily with either Met-RANTES or vehicle starting on postoperative day 4 and were euthanized on postoperative days 24 and 56. We found that Met-RANTES significantly reduced intimal thickening in this model of chronic rejection and that Met-RANTES markedly decreased the infiltration of CD4 and CD8 T lymphocytes and MOMA-2 ϩ monocytes/macrophages into transplanted hearts. Met-RANTES also suppressed the ex vivo and in vitro proliferative responses of recipient splenocytes to donor antigens. Finally, Met-RANTES treatment was associated with a marked reduction in RANTES/CCL5 and monocyte chemoattractant protein-1 gene transcript levels in the donor hearts. Conclusions-Antagonism of the chemokine receptors CCR1 and CCR5 with Met-RANTES attenuates CAV development in vivo by reducing mononuclear cell recruitment to the transplanted heart, proliferative responses to donor antigens, and intragraft RANTES/CCL5 and monocyte chemoattractant protein-1 gene transcript levels. These findings suggest that chemokine receptors CCR1 and CCR5 play significant roles in the development of chronic rejection and may serve as potential therapeutic targets.
Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.
The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.
DNA double strand breaks are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure, with high affinity for double strand breaks. It relocates to sites of damage independently of signalling and known sensors and activates downstream signalling cascades for double strand break repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the Homologous Recombination and Non-Homologous End Joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as DNA damage sensor, which is critical for initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB binding capacity and DDR activation. SIRT6 activates the DDR, before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 at the top of the DDR and pave the road to dissect the contributions of distinct double strand break sensors in downstream signalling.
Many pharmacologically important agents are assembled on multimodular nonribosomal peptide synthetases (NRPSs) whose modules comprise a set of core domains with all essential catalytic functions necessary for the incorporation and modification of one building block. Very often, d‐amino acids are found in such products which, with few exceptions, are generated by the action of NRPS integrated epimerization (E) domains that alter the stereochemistry of the corresponding peptidyl carrier protein (PCP) bound l‐intermediate. In this study we present a quantitative investigation of substrate specificity of four different E domains (two ‘peptidyl‐’ and two ‘aminoacyl‐’E domains) derived from different NRPSs towards PCP bound peptides. The respective PCP‐E bidomain apo‐proteins (TycB3‐, FenD2‐, TycA‐ and GrsA‐PCP‐E) were primed with various peptidyl‐CoA precursors by utilizing the promiscuous phosphopantetheinyl transferase Sfp. PCP bound peptidyl‐S‐Ppant epimerization products were chemically cleaved and analyzed for their l/d‐ratios by LCMS. We were able to show that all four E domains tolerate a broad variety of peptidyl‐S‐Ppant‐substrates as evaluated by kobs values and final l/d‐product equilibria determined for each reaction. The two C‐terminal amino acids of the substrate seem to be recognized by ‘peptidyl‐’E domains. Interestingly, the ‘aminoacyl‐’E domains GrsA‐ and TycA‐E were also able to convert the elongated intermediates. All four E domains accepted an N‐methylated precursor as well and epimerized this substrate with high efficiency. Finally, we could demonstrate that the condensation (C) domain of TycB1 is also able to process peptidyl substrates transferred by TycA. In conclusion, these findings are of great impact on future engineering attempts.
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