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    • The observed severe inhibition in MM cells is

    2018-10-30

    The observed severe inhibition in MM nicergoline cost is surprising, because lysing the cells prior to treatment with the same concentration of bortezomib only resulted in a 40% reduction in activity. After eliminating potential trivial explanations, the authors reach the conclusion that specifically in living cells proteasome inhibitor treatment results in some indirect enhanced inhibition. The authors identify changes in a post-translational modification for several proteasome subunits. This modification, which remains to be fully characterized, has previously been reported by the same group (). It appears to be mainly nuclear, have unusual biochemical properties, and similarities to ADP ribosylation. As the modification remains somewhat elusive, manipulating the levels of modification to determine its effect on proteasome activity is difficult. Nevertheless, the authors show that a specific treatment (venom phosphodiesterase-1 with S1 nuclease) changes the modification on some proteasome subunits in vitro. This change correlated with reduced proteasome activity. Summarizing these results, it suggests that treating cells with proteasome inhibitor has two effects. First, there is a direct inhibition resulting from inactivation of proteasome active sites. Second, there is an indirect inhibition, where bortezomib induces post-translational modifications on the proteasome that reduce proteasome activity. This intriguing new model raises a whole set of new questions. What is the identity of the post-translational modification and what is the enzyme responsible? Are these more abundant in MM cells as compared with cells that are more resistant to bortezomib treatment? Identifying them would allow researchers to rigorously test the model by showing that cells are less sensitive to bortezomib upon elimination of the modification. Furthermore, it will allow researchers to test if this inhibitory proteasome modification is induced or increased specifically in cells sensitive for proteasome inhibitors. From a biochemical perspective, it is interesting that the presence of a proteasome inhibitor buried within the large enzyme can result in activation of an enzyme responsible for post-translational modifications. It has previously been shown that proteasome inhibitors induce structural changes in the proteasome (). These changes could trigger the recognition by enzymes responsible for the post-translational modification; however, this link between structural changes and induction of post-translational modifications remains to be shown. One problem with bortezomib treatment in the clinic is the occurrence of drug resistance, for example through mutation in the bortezomib binding subunit PSMD5 (). However, not all causes of resistance have been determined. If patients acquire mutations that interfere with the ability to induce indirect inhibition as described here (), it would be predicted to cause resistance to bortezomib. Besides bortezomib, there are new proteasome inhibitor drugs under development, like the FDA-approved second generation proteasome inhibitor carfilzomib, for treating bortezomib-resistant patients and patients experiencing severe bortezomib-related side effects and toxicity (). It will be important to determine the extent to which the phenomena described by Pitcher et al. are specific for bortezomib, chymo-trypsin-like inhibitors, or all proteasome inhibitors.
    Spliceosome-mediated RNA trans-splicing (SMaRT) is a molecular tool that facilitates genetic reprogramming on the RNA level (). SMaRT exploits the cells own splicing machinery to recombine two RNA molecules: the endogenous RNA target and the RNA trans-splicing molecule (RTM). The end product is a chimeric RNA wherein part of the message encoded by the target RNA is replaced with one provided by the RTM. The specificity of the trans-splicing reaction is conferred by an anti-sense binding domain complementary to a non-coding region within the target RNA. This serves to tether the RTM to its target RNA, bringing donor and acceptor splice sites on both molecules into close proximity thereby allowing the trans-splicing reaction to occur. Since its first demonstration in 1999, where it was used in a cancer suicide gene therapy approach (), SMaRT has primarily made a niche in the field of gene therapy where it has successfully been employed in pre-clinical investigations for the therapy of various genetic disorders, including cystic fibrosis, hemophilia A, spinal muscular atrophy, and the severe skin blistering disease epidermolysis bullosa (as reviewed in ).