Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

BACKGROUND: Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. RESULTS: While identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA. CONCLUSION: Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death

Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

Abstract Background Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. Results While identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA. Conclusion Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.</p

Biodegradable poly(ester amine) based on glycerol dimethacrylate and polyethylenimine as a gene carrier

Background Polyethylenimine (PEI) vectors are widely used in gene delivery because of their high transfection efficiency owing to a unique proton sponge effect. An increase in molecular weight increases transfection efficiency, but simultaneously results in increased toxicity. Therefore, the design and synthesis of new degradable gene delivery carriers having high transfection efficiencies and reduced cytotoxicity are necessary. Methods in the present study degradable poly(ester amines) (PEAS) based on glycerol dimethacrylate (GDM) and low molecular weight branched polyethylenimine (LMW-PEI) were synthesized in anhydrous methanol at 60 degrees C following Michael addition reaction. The transfection efficiencies of the synthesized PEA/DNA complexes were evaluated using three different cell lines (HeLa, HepG2 and 293T cells) in vitro. Results PEAS with zeta potential in the range of 30-55 mV (at physiological pH) condensed plasmid DNA into nanosized particles (< 150 nm) suitable for intracellular delivery. The PEAS degraded in a controlled fashion (t(1/2) of approximately 9-10 days). Compared with PEI 25K, the PEAS showed significantly lower cytotoxicity in three different cells. The PEAS demonstrated much higher transfection efficiency compared to conventional PEI 25K and Lipofectamine. The PEA synthesized using a 1: 4 mole ratio of GDM to PEI [GDM/PEI-1.2 (1 : 4)] showed the highest transfection efficiency in HepG2 cells. Significantly higher pEGFP-N-2 reporter gene expression in 293T cells was achieved using these PEAS. The hyperosmotic effect of PEAS was demonstrated by the reduction in packed cell volume (PCV). The GDM/PEI-1.2 (1 : 4) showed comparable reduction in PCV with respect to glycerol in 293T cells. The effect of bafilomycin A, on transfection efficiency of PEAS on 293T cells indicated its endosomal buffering capacity. Conclusions We hypothesized that the higher transfection efficiency of PEAS was the synergistic effect arising from hyperosmotic glycerol and endosomal buffering capacity of PEAS resulting from the presence of a glycerol backbone and PEI amine groups, respectively. Copyright (C) 2008 John Wiley & Sons, Ltd