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Dehydrating stresses trigger the accumulation of abscisic acid (ABA), a key plant stresssignaling hormone that activates Snf1-Related Kinases (SnRK2s) to mount adaptive responses.However, the regulatory circuits that terminate the SnRK2s signal relay after acclimation or poststress conditions remain to be defined. Here, we show that the desensitization of the ABA-signal is achieved by the regulation of OST1 (SnRK2.6) protein stability via the E3-ubiquitin-ligase HOS15. Upon ABA signal, HOS15-induced degradation of OST1 is inhibited and stabilized OST1 promotes the stress-response. When the ABA signal terminates, protein phosphatases ABI1/2 recruit HOS15 to OST1 to promote the rapid degradation of OST1. Notably, we found that even in the presence of ABA, OST1 levels were also depleted within hours of ABA signal onset. The unexpected dynamics of OST1 abundance was resolved by a systematic mathematical modeling demonstrating a desensitizing feedback loop by which OST1-induced up-regulation of ABI1/2 leads to the degradation of OST1. This model illustrates the complex rheostat dynamics underlying the ABA-induced stress response and desensitization. signaling components. Several members of the PYR/PYL/RCAR family of ABA receptors are specifically recognized by different E3 ubiquitin ligases and targeted for degradation through proteasome action (Irigoyen et al., 2014). ABI1, a PP2C phosphatase that inhibits ABA-related SnRK2 kinases such as OST1, is ubiquitinated by PUB12/PUB13 (U-box E3 ligases) and also degraded by the proteasome in the presence of ABA signal (Kong et al., 2015), which then facilitates the activation of SnRK2 kinases and of their downstream transcription factors (TFs).Eventually, the TFs that accumulate in response to ABA need to be degraded when the signal ceases. When ABA signaling stops, ABI FIVE BINDING PROTEIN1 (AFP1) and KEG (KEEP ON GOING) facilitate UPS-mediated proteolysis of ABI5 and ABF1/ABF3 (Lopez-Molina et al., 2003;Stone et al., 2006;Chen et al., 2013;Liu et al., 2013). In addition, DWA1/DWA2 (DWD HYPERSENSITIVE TO ABA1/2), and ABD1 (ABA-HYPERSENSITIVE DCAF1), substrate receptors for the DDB1 CULLIN4-based E3 ligases, command the degradation of ABI5 (Seo et al., 2014;Lee et al., 2010). The positive signaling effectors SnRK2.2, SnRK2.3 and SnRK2.6/OST1 are known to be degraded by an ubiquitination-and proteasome-dependent mechanism, but the mechanism involved has not been identified with the exception of SnKR2.3 that was shown to be degraded by AtPP2-B11 (Kim et al., 2013;Cheng et al., 2017). In summary, the degradation of positive signaling effectors leads to deactivation of the ABA signal pathway.The ubiquitin-26S proteasome system (UPS) proceeds via sequential reactions performed by three distinct sets of enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin protein ligases (E3). Because target specificity is conferred by the E3 ligases, plant genomes encode hundreds of E3 ligases that recruit specific target proteins in multiple biological pro...

A series of eight bis(thiosemicarbazone) ligands and 16 of their respective copper(II) and zinc(II) complexes containing a combination of hydrogen, methyl, pyridyl, phenyl, and/or ethyl substituents at the diimine position of the ligand backbone were synthesized and characterized. The objective of this study was to identify the structure−activity relationships within a series of analogues with different substituents at the diimine position of the backbone and at the terminal N atom. The Cu(II) complexes Cu(GTSM 2 ), Cu(GTSCM), Cu-(PyTSM 2 ), Cu(EMTSM 2 ) and Cu(PGTSM 2 ) demonstrated a distorted square planar geometry, while the Zn(II) complexes Zn(ATSM 2 )(DMSO), Zn(PyTSM 2 )(DMSO), and Zn-(PGTSM 2 )(H 2 O) formed a distorted square pyramidal geometry. Cyclic voltammetry showed that the Cu(II) complexes display quasi-reversible electrochemistry. Of the agents, Cu(II) glyoxal bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(GTSM 2 )] and Cu(II) diacetyl bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(ATSM 2 )] demonstrated the greatest antiproliferative activity against tumor cells. Substitutions at the diimine position and at the terminal N atom with hydrophobic moieties markedly decreased their antiproliferative activity. Complexation of the bis(thiosemicarbazones) with Zn(II) generally decreased their antiproliferative activity, suggesting the Zn(II) complex did not act as a chaperone to deliver the ligand intracellularly, in contrast to similar bis(thiosemicarbazone) cobalt(III) complexes [King et al. Inorg. Chem. 2017, 56, 6609−6623]. However, five of the eight bis(thiosemicarbazone) Cu(II) complexes maintained or increased their antiproliferative activity, relative to the ligand alone, and a mechanism of Cu-induced oxidative stress is suggested. Surprisingly, relative to normoxic growth conditions, hypoxia that is found in the tumor microenvironment decreased the antiproliferative efficacy of most bis(thiosemicarbazones) and their copper complexes. This was independent of the potential hypoxia−selectivity mediated by Cu(II/I) redox potentials. These results provide structure−activity relationships useful for the rational design of bis(thiosemicarbazone) anticancer agents.

In memory of Rainer RudolphBreast cancer is still one of the most frequently occurring tumors in women. Severe and often therapy-limiting side effects are a major obstacle in chemotherapy. New delivery concepts that reduce systemic side effects are needed to optimize anticancer therapies, and selective targeting concepts are required for early and selective tumor diagnosis. Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is a C-terminal amidated peptide hormone consisting of 36 amino acid residues.[1, 2] NPY-mediated functions are transmitted by so-called Y receptors, named Y 1 , Y 2 , and Y 5 receptors, which bind NPY with nanomolar affinity. All Y receptors are members of the class A of heptahelix receptors, that signal through heterotrimeric G proteins. [3,4] Reubi et al. have recently described Y-receptor expression in human breast cancer. They have shown that over 90 % of all breast tumors and 100 % of the examined metastases express Y 1 receptors.[5] Interestingly, a shift of the receptor subtype from Y 2 receptors in healthy tissue to Y 1 receptors during neoplasm was found, which is potentially related to reduced differentiation. Based on NPY and the known structure-activity relationships for Y 1 -receptor binding, [6] we designed, synthesized, and characterized two analogues for tumor labeling that vary in the position of the chelator to conjugate 99m Tc. Peptides 1 a and 2 a were synthesized with a N a -histidinyl acetyl (N a His-ac) chelator [7] at the N terminus, whereas peptides 1 b and 2 b were modified at the N e side chain of Lys 4 . The tridentate ligand N a His-ac is able to form stable and biologically active complexes. [8,9] Modification of the resin-bound peptide was performed by an efficient strategy (Scheme 1). In the first step, bromoacetic acid was activated by diisopropylcarbodiimide to form the corresponding anhydride. His(Trt)-OtBu was then added and the NHÀ CH bond was formed by HBr elimination. Cleavage of the peptide yielded His-acetyl peptides either at the N terminus or at the N e side chain of Lys 4 . Rhenium was used as a cold surrogate for 99m Tc and introduced for in vitro studies