Effects of chronic tramadol exposure on the zebrafish brain: a proteomic study

Tramadol hydrochloride (TH), has become the most prescribed opioid worldwide. However, its neurotoxicity and abuse potential are not well documented. In the present study, TH administration induced abnormal behavior and body and brain mean weight loss. Two principal metabolites O- and N-desmethyltramadol were detected in the brain tissue, and N-desmethyltramadol was the main metabolite produced. A total of 30 differential protein spots were identified using semi-quantitative 2D-PAGE and proteomic analyses, and classified into 13 categories, in which subtypes of 14-3-3 proteins, creatine kinase, ATP synthase beta chain, and tubulin were identified at the separated location on the gels 3, 3, 4, and 11 times respectively. Many TH responsive proteins have functions related to oxidative stress, including 14-3-3 proteins, creatine kinase BB, ubiquitin carboxy-terminal hydrolase L-1, ATP synthase, synaptosome-associated protein, tubulin and actin. Irrespective of oxidative damage, other pathways affected include apoptosis, energy metabolism, signal disorders, and cytoskeletal structure. Ultrastructural observation of mitochondria showed a series of morphological changes in the case of TH exposure.     

Phosphorylation screening identifies translational initiation factor 4GII as an intracellular target of Ca(2+)/calmodulin-dependent protein kinase I

CaMKI is a Ca2+/calmodulin-dependent protein kinase that is widely expressed in eukaryotic cells and tissues but for which few, if any, physiological substrates are known. We screened a human lung cDNA expression library for potential CaMKI substrates by solid phase in situ phosphorylation ("phosphorylation screening"). Multiple overlapping partial length cDNAs encoding three proteins were detected. Two of these proteins are known: 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase and eukaryotic translation initiation factor (eIF) 4GII. To determine whether CaMKI substrates identified by phosphorylation screening represent authentic physiological targets, we examined the potential for [Ca2+]i- and CaMKI-dependent phosphorylation of eIF4GII in vitro and in vivo. Endogenous eIF4GII immunoprecipitated from HEK293T cells was phosphorylated by CaMKI, in vitro as was a recombinant fragment of eIF4GII encompassing the central and C-terminal regions. The latter phosphorylation occurred with favorable kinetics (Km = 1 microm; kcat = 1.8 s-1) at a single site, Ser1156, located in a segment of eIF4GII aligning with the phosphoregion of eIF4GI. Phosphopeptide mapping and back phosphorylation experiments revealed [Ca2+]i-dependent, CaMKI site-specific, eIF4GII phosphorylation in vivo. This phosphorylation was blocked by kinase-negative CaMKI consistent with a requirement for endogenous CaMKI for in vivo eIF4GII phosphorylation. We conclude that phosphorylation screening is an effective method for searching for intracellular targets of CaMKI and may have identified a new role of Ca2+ signaling to the translation apparatus.     

Design and synthesis of diarylamines and diarylethers as cytotoxic antitumor agents

Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cytotoxic hit 6-chloro-2-(4-cyanophenyl)amino-3-nitropyridine (7), 30 diarylamines and diarylethers were designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI(50) values ranging from 0.33 to 3.45μM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring) were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential biological targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and 13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types, with IC(50) values of 2.2-3.0μM. Therefore, these findings provide a good starting point to optimize a new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.     

The regulation of necroptosis by ubiquitylation

Necroptosis is a programmed necrosis that is mediated by receptor-interacting protein kinases RIPK1, RIPK3 and the mixed lineage kinase domain-like protein, MLKL. Necroptosis must be strictly regulated to maintain normal tissue homeostasis, and dysregulation of necroptosis leads to the development of various inflammatory, infectious, and degenerative diseases. Ubiquitylation is a widespread post-translational modification that is essential for balancing numerous physiological processes. Over the past decade, considerable progress has been made in the understanding of the role of ubiquitylation in regulating necroptosis. Here, we will discuss the regulatory functions of ubiquitylation in necroptosis signaling pathway. An enhanced understanding of the ubiquitylation enzymes and regulatory proteins in necroptotic signaling pathway will be exploited for the development of new therapeutic strategies for necroptosis-related diseases.