AMN-107 The expression of cytokine inducible genes is mediat
The expression of cytokine-inducible genes is mediated by a number of components within the cell. It has been shown that Jak2 and the Ras/Raf/MAP kinase signaling cascades are required for the expression of DUB-1, . However, the enhancer domain of DUB-1 lacks a consensus sequence for Stat binding, leading to the investigation of cytokine inducible enhancer activity of the 5′ flanking region . Previous investigations indicated that two AP-1 sites and a GATA motif at the N-terminus, in addition to a consensus sequence for Ets binding, are major cytokine inducible elements of the DUB-1 gene . In this study, we found that the enhancer sequence of DUB-2A is highly homologous to that of DUB-1, and showed important roles of two AP-1 sites and an Ets site for cytokine inducibility (Fig. 4), suggesting regulatory conservation for their sequences. Even though the biological function of Dub-2A enzyme is not understood, it has been suggested that Dub-2A may play a role in the regulation of hematopoietic cell growth in T-lymphocytes as does Dub-1 in B-lymphocytes . Finding substrates and the molecular mechanism of deubiquitination by these AMN-107 will clarify to understand their biological roles in regulating the status of protein degradation.
Taken together, finding accelerators or inhibitors of catalytic activity for the Dub-2A deubiquitinating enzyme and of DUB-2A expression will help to regulate their cellular functions in T-lymphocytes.
Introduction The ubiquitin–proteasome pathway (UPP) plays a vital role in the degradation of proteins involved in several pathways, including cellular proliferation and apoptosis. The proteasome is a validated target for multiple myeloma (MM) treatment; proteasomal inhibitors form a cornerstone of anti-myeloma therapy . These inhibitors include bortezomib (PS-341), the first anti-MM proteasomal inhibitor that was FDA-approved, in 2003. Carfilzomib (Kyprolis), an epoxyketone with specific chymotrypsin-like activity, acts as an irreversible proteasomal inhibitor and was approved by the FDA in 2012 due to the improved response observed in relapsed and refractory MM patients previously treated with bortezomib . However, in spite of its improved efficacy compared to alternative therapies, approximately 60% of patients do not respond to bortezomib due to the emergence of resistance. In addition to proteasome, the UPP also includes ubiquitin, ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s) and deubiquitinases (DUBs), which collectively work in three discrete and successive steps: 1) the substrate is tagged by the covalent attachment of multiple ubiquitin molecules via E1, E2, and E3, 2) the tagged protein is degraded by the 26S proteasomal complex, and 3) poly-ubiquitins are recycled by DUBs to free ubiquitin for reuse . Multiple myeloma is the most sensitive and the most responsive disease to proteasomal inhibitors, which implies that the UPP is critical for multiple myeloma pathophysiology. E1 , E2s such as CDC34 , E3s such as Mdm-2  and SCF , and DUBs such as USP9X  are overexpressed and involved in multiple myeloma pathology. Furthermore, targeting such UPP components could sensitize MM cells susceptible to bortezomib-induced cell death , . Thus, targeting the entire UPP as opposed to proteasome alone is a promising strategy for multiple myeloma treatment. It has been shown that the functional analysis of the UPP can be accomplished by following the steady-state levels of GFP reporter substrates, which are typically based on an intrinsic fluorescent protein with a constitutively active degradation signal that targets the proteins for ubiquitination and proteasomal degradation . Such a reporter system could be used to find new UPP inhibitors with distinct mechanisms and novel structures . Here, the natural compound curcusone D, a diterpene isolated from Jatropha curcas (Barbados nut), an herbal plant that has been used in traditional folk medicine in many tropical countries, was identified to be a novel UPP inhibitor with the Ub-G76V-YFP reporter assay. The purpose of the present study was to investigate the mechanism of UPP inhibition by curcusone D and to further characterize its anti-MM effects. Curcusone D could not inhibit the proteasome's chymotrypsin-like, trypsin-like or caspase-like activities in vitro or at the cellular level, but it did inhibit the activity of DUBs in cells. Further mechanistic studies showed that curcusone D could induce ROS, which are responsible for the inhibition of DUBs and the UPP to induce cellular growth inhibition and apoptosis. Curcusone D could inhibit multiple myeloma cell growth and induce cellular apoptosis. The combination of curcusone D with bortezomib had significant synergistic effects on MM cell growth inhibition, apoptosis and UPP inhibition.