In the presence of non-stoichiometric amounts of proteasome CP, Pup-GGQ is completely bound to Msm Mpa with the C terminus binding to the mouth of the hexamer and the Nterminus falling into the central cavity. The charge difference between glutamine and glutamate in Pup may affect proteasomal degradation. In conclusion, we assembled a functional 1.2 megadalton mycobacterium proteasome, consisting of Mpa and the proteasome CP, inside E. coli to study its interactions with the prokaryotic ubiquitin-like protein, Pup-GGQ at amino acid residue resolution. By using STINT-NMR, we show that in-cell proteasomal degradation is dynamically regulated by transient interactions between Mpa and the proteasome CP. Differences between the binding of Pup and Mpa in vivo and in vitro underscore the importance of studying interactions under close to physiological conditions. Oncolytic viruses have been proposed for cancer therapy, since they can be engineered to potentially deliver their cytocidal effect to tumor cells. Among them, adenoviruses were widely used as oncolytic viral agents in cancer therapy, as they possess an inherent potential to kill the cells that sustain their replication. However, to restrict cytocidal effect to tumor cells, their replication had to be tightly controlled in normal cells. Hence, conditionally replicative adenoviruses have been developed to restrict viral replication to target FDA-approved Compound Library cancerous tissues and inhibit replication in normal healthy cells. This has been attempted by exploiting loss-offunction mutations in E1B viral sequences, or linking genes E1A/E1B to cancer-specific promoters, such as the telomerase or prostate-specific rat probasin promoters or the human prostate-specific enhancer/promoter. Additional approaches have also been explored for specific targeting viruses to cancer cells, for example through the introduction of T-cell receptors specific for tumor-specific antigens. More recently, a strategy based on endogenous microRNAs has been explored to control viral replication. miRNAs are small, non-coding RNA molecules of 20�C24 bp that can regulate gene expression at the post-transcriptional level by binding to target transcripts in a sequence-specific manner. These small molecules play a critical role in many cellular processes and several examples of aberrantly regulated miRNAs in human cancer have been reported. Among all, miRNAs expression profiling in hepatocellular carcinomas revealed the existence of differential patterns between tumor tissues and normal liver. In particular, miR-199 was reported to be consistently down-regulated in HCC. The involvement of miR-199 in the pathogenesis of HCC was linked to the abnormal regulation of multiple target genes, such as mTOR, c-Met, HIF-1�� and CD44. Here, we took advantage of this information to produce a new type of CRAd able to replicate only in cells lacking miR-199, with the aim of making viral replication and cytolytic effect specifically selective for HCC cells. HCC, the fifth most frequent neoplasm and the third leading cause of cancer-related deaths worldwide, carries a generally poor prognosis. Complete tumor removal represents the only long-term cure. However, partial hepatectomy can be undertaken in less than 15-30% of patients due to the extent of underlying cirrhosis and, among patients who undergo tumor resection, up to 75% of patients will develop intra-hepatic recurrences within 5 years. When possible, complete hepatectomy and orthotopic liver transplantation represents the therapy of choice for patients with significant cirrhosis and limited tumor MK-0683 burden. In patients who are not candidates for liver transplantation or resection, the most common therapy is transcatheter arterial chemoembolization, whose impact on clinical outcome remains unclear. The use of systemic chemotherapy has been attempted but HCC is minimally responsive.