Simultaneous quantification of total and conjugated ubiquitin levels in a single immunoblot.

Ubiquitin (Ub) is a posttranslational modifier, and total Ub (UbT) is always in dynamic equilibrium among free Ub (UbF), activated Ub (UbA), and conjugated Ub (UbC) in the forms of mono-Ub, thioester-bond-linked Ub, and peptide-bond-linked Ub, respectively. In this study, we developed a simple method to simultaneously determine the levels of UbT, UbF+UbA, and UbC in a single immunoblot and demonstrated its reliability and reproducibility by determining [UbT], [UbF+UbA], and [UbC] in various mouse tissues and cultured cells.

Large-scale production of soluble recombinant amyloid-ß peptide 1-42 using cold-inducible expression system.

Amyloid-ß peptide 1-42 (Aß(1-42)), the predominant form in senile plaques, plays important roles in the pathogenesis of Alzheimer's disease. Because Aß(1-42) has aggregation-prone nature, it has been difficult to produce in a soluble state in bacterial expression systems. In this study, we modified our expression system to increase the soluble fraction of Aß(1-42) in Escherichia coli (E. coli) cells. The expression level and solubility of recombinant Aß(1-42) induced at the low temperature (16°C) is highly increased compared to that induced at 37°C. To optimize expression temperature, the coding region of Aß(1-42) was constructed in a pCold vector, pCold-TF, which has a hexahistidine-tagged trigger factor (TF). Recombinant Aß(1-42) was expressed primarily as a soluble protein using pCold vector system and purified with a nickel-chelating resin. When the toxic effect of recombinant Aß(1-42) examined on human neuroblastoma SH-SY5Y cells, the purified Aß(1-42) induced cell toxicity on SH-SY5Y cells. In conclusion, the system developed in this study will provide a useful method for the production of aggregation prone-peptide such as Aß(1-42).

Functional differences between two major ubiquitin receptors in the proteasome; S5a and hRpn13.

It is well known that S5a and hRpn13 are two major ubiquitin (Ub) receptors in the proteasome but little is known about their functional difference in recruiting ubiquitinated substrates. In this study using siRNA-mediated knockdown of S5a or hRpn13, we found that two Ub receptors had different substrate specificity although similar level of accumulation of high molecular weight Ub-conjugates was observed. Interesting enough, depletion of S5a, but not hRpn13, resulted in the Ub-containing aggregates and induced ER chaperones such as Grp78 and Grp94. ERAD substrates such as alpha-TCR and alpha1-antitrypsin were also stabilized by the depletion of S5a but not hRpn13. Our results suggest that there is different substrate specificity between S5a and hRpn13 at the level of delivery and S5a may be the major docking site for ERAD substrates.

Scientific rationale behind the development and approval of biosimilar infliximab (CT-P13) in Europe.

Biosimilars are drugs developed to be highly similar to their originator biologic (or 'reference medicinal product') with no clinically meaningful differences in purity, efficacy or safety. Production of biologics and biosimilars is highly complex and sensitive, with any change in manufacturing process having a potential impact on efficacy and safety. This review provides an overview of the manufacturing process for these drugs and considers the implications of any process changes. The scientific rationale underlying the regulatory comparability exercise for process-changed reference medicinal products and biosimilars is also discussed, as is the issue of 'switchability' from a reference medicinal product to its biosimilar. CT-P13 (Remsima(®), Inflectra(®)), a biosimilar of infliximab, is used as a case study to discuss these issues.

Analysis of clinical trials of biosimilar infliximab (CT-P13) and comparison against historical clinical studies with the infliximab reference medicinal product.

OBJECTIVE: To examine whether efficacy, safety and pharmacokinetic (PK) data observed with CT-P13 (Remsima(®); Inflectra(®)), an infliximab biosimilar, are similar to those from published reports with the reference medicinal product (RMP; Remicade(®)) in patients with rheumatoid arthritis (RA) or ankylosing spondylitis (AS). METHODS: Literature searches were performed to identify clinical studies with infliximab RMP. Efficacy, safety and PK data were indirectly compared with data from head-to-head clinical trials of CT-P13 and RMP. RESULTS: CT-P13 and RMP produce similar efficacy in patients with RA and AS when compared across clinical studies. There are no substantial differences in the incidence of infusion-related reactions, infections, serious infections, malignancy or lymphoma. PK data in patients with RA are similar in direct comparisons and comparisons with historical data. CONCLUSION: Efficacy, safety and PK data are highly comparable between CT-P13 and RMP, both in head-to-head clinical studies, and indirect comparisons with historical clinical data for RMP.

Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention.

Ubiquitin is involved in almost every cellular process, and it is also known to be a stress-inducible protein. Based on previous reports that many types of cancer display an elevated level of ubiquitin, we hypothesized that this increased amount of ubiquitin is essential for the growth of cancer cells and that, consequently, the downregulation of ubiquitin may be a potential anti-cancer treatment. We first found that the level of ubiquitin can be effectively downregulated via knockdown of a polyubiquitin gene, Ubb, with siRNA (Ubb-KD) and then demonstrated its anti-cancer effects in several cancer cell lines and xenograft mice. Ubb-KD resulted in the attenuation of TNFα-induced NF-κB activation, the stabilization of the tumor suppressor p53, and stress-sensitization. Taken together, downregulation of ubiquitin through Ubb-KD is a potential anti-cancer treatment by inhibiting ubiquitination at multiple sites related to oncogenic pathways and by weakening the ability of cancer cells to overcome increased stress.