Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green florescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar K M values, but SUMO protease had a 25-fold higher k cat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.

Tagging proteins with mono-or poly-ubiquitin is now recognized as a multifaceted and universal means of regulating cell growth and physiology. It does so by controlling the cellular lifetime of nearly all eukaryotic proteins and the cellular localization of many critical proteins. Enzymes of the ubiquitin pathway add (ligases) or remove (deubiquitinases [DUBs]) ubiquitin tags to or from their target proteins in a selective fashion. Similarly to the kinases and their corresponding phosphatases, ubiquitin ligases and DUBs have become actively studied molecular oncology targets for drug discovery. Approximately 79 functional DUBs exist in the human proteome, suggesting that selective intervention is a reasonable therapeutic objective, with the goal of downregulating or ablating oncogene products or, alternatively, upregulating or sparing tumor suppressors. In the following review, this fascinating class of regulatory enzymes will be described, and specific examples of DUBs that are viable targets for anticancer therapy will be considered. Keywordsassociated molecule with the SH3-domain of STAM; cylindromatosis gene; deubiquitinating enzymes; isopeptidase; proteasomal degradation; ubiquitin; ubiquitin-specific protease 2a; ubiquitinspecific protease 7; ubiquitin-specific protease 20 Ubiquitin & ubiquitin-like proteinsThe content of most proteins in the cell is regulated by the ubiquitin-proteasomal pathway [1]. Ubiquitin and ubiquitin-like proteins (UBLs), such as SUMO, NEDD8, ISG15 and FAT10, regulate proteins via additional mechanisms, for example, intracellular compartmentation, signal transduction and the regulation of some E3 ligases [2]. Degradation of a targeted protein by the ubiquitin system involves the activation of ubiquitin by the enzyme E1, which links the ubiquitin C-terminus to a cysteine side chain of the enzyme in an ATP-dependent manner [1]. Activated ubiquitin is transferred as a thioester to enzyme E2, which catalyzes ubiquitin †Author for correspondence: Progenra, Inc., 271A Great Valley Parkway,

Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.

Dysregulation of the ubiquitin-proteasome system (UPS) has been implicated in a wide range of pathologies including cancer, neurodegeneration, and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; yet toxicity with this target remains high. Ubiquitin Ligases (E3s) represent an alternative attractive therapeutic target in the UPS. Here we will discuss current platforms that report on E3 ligase activity and can detect E3 inhibitors, while underlining the advantages and disadvantages of each approach.

The ubiquitin pathway regulates diverse functions including protein localization and stability. The complexity of the pathway involving nearly 40 identified E2 conjugating enzymes and over 600 E3 ligases raises the issue of specificity. With the E2s and E3s fitting into a limited number of classes based on bioinformatics, structures, and proven activities, there is not a clear picture as to what would determine which E2/E3 enzyme pair would be functional. There have been many reports of limited E2/E3 activity profiling with a small number of E2s and E3s. We have expanded on this to investigate the activity of ubiquitin E2s covering the majority of the reported classes/families in concert with a number of E3s implicated in a variety of diseases. Using an ELISA-based assay we screened 10 E3 ligases against a panel of 11 E2s to determine which E2/E3 pairs exhibited E3 autoubiquitylation activity. In addition, the ubiquitin chain linkage preference by certain E2/E3 pairs was investigated. Finally, substrate ubiquitylation was assayed for the E3 ligase MuRF1 using various E2/MuRF1 pairs. These studies demonstrate the utility of identifying the correct E2/E3 pair to monitor specific substrate ubiquitylation.