GNIP1 E3 ubiquitin ligase is a novel player in regulating glycogen metabolism in skeletal muscle

BACKGROUND: Glycogenin-interacting protein 1 (GNIP1) is a tripartite motif (TRIM) protein with E3 ubiquitin ligase activity that interacts with glycogenin. These data suggest that GNIP1 could play a major role in the control of glycogen metabolism. However, direct evidence based on functional analysis remains to be obtained. OBJECTIVES: The aim of this study was 1) to define the expression pattern of glycogenin-interacting protein/Tripartite motif containing protein 7 (GNIP/TRIM7) isoforms in humans, 2) to test their ubiquitin E3 ligase activity, and 3) to analyze the functional effects of GNIP1 on muscle glucose/glycogen metabolism both in human cultured cells and in vivo in mice. RESULTS: We show that GNIP1 was the most abundant GNIP/TRIM7 isoform in human skeletal muscle, whereas in cardiac muscle only TRIM7 was expressed. GNIP1 and TRIM7 had autoubiquitination activity in vitro and were localized in the Golgi apparatus and cytosol respectively in LHCN-M2 myoblasts. GNIP1 overexpression increased glucose uptake in LHCN-M2 myotubes. Overexpression of GNIP1 in mouse muscle in vivo increased glycogen content, glycogen synthase (GS) activity and phospho-GSK-3alpha/beta (Ser21/9) and phospho-Akt (Ser473) content, whereas decreased GS phosphorylation in Ser640. These modifications led to decreased blood glucose levels, lactate levels and body weight, without changing whole-body insulin or glucose tolerance in mouse. CONCLUSION: GNIP1 is an ubiquitin ligase with a markedly glycogenic effect in skeletal muscle

Several E4 Region Functions Influence Mammary Tumorigenesis by Human Adenovirus Type 9

Among oncogenic adenoviruses, human adenovirus type 9 (Ad9) is unique in eliciting exclusively estrogen-dependent mammary tumors in rats and in not requiring viral E1 region transforming genes for tumorigenicity. Instead, studies with hybrid viruses generated between Ad9 and the closely related nontumorigenic virus Ad26 have roughly localized an Ad9 oncogenic determinant(s) to a segment of the viral E4 region containing open reading frame 1 (E4-ORF1), E4-ORF2, and part of E4-ORF3. Although subsequent findings have shown that E4-ORF1 codes for an oncoprotein essential for tumorigenesis by Ad9, it is not known whether other E4 region functions may similarly play a role in this process. We report here that new results with Ad9/Ad26 hybrid viruses demonstrated that the minimal essential Ad9 E4-region DNA sequences include portions of both E4-ORF1 and E4-ORF2. Investigations with Ad9 mutant viruses additionally showed that the E4-ORF1 protein and certain E4-ORF2 DNA sequences are necessary for Ad9-induced tumorigenesis, whereas the E4-ORF2 and E4-ORF3 proteins are not. In fact, the E4-ORF3 protein was found to antagonize this process. Also pertinent was that certain crucial nucleotide differences between Ad9 and Ad26 within E4-ORF1 and E4-ORF2 were found to be silent with respect to the amino acid sequences of the corresponding proteins. Furthermore, supporting a prominent role for the E4-ORF1 oncoprotein in Ad9-induced tumorigenesis, an E1 region-deficient Ad5 vector that expresses the Ad9 but not the Ad26 E4-ORF1 protein was tumorigenic in rats and, like Ad9, promoted solely mammary tumors. These findings argue that the E4-ORF1 oncoprotein is the major oncogenic determinant of Ad9 and that an undefined regulatory element(s) within the E4 region represents a previously unidentified second function likewise necessary for tumorigenesis by this virus