Abstract
Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13−/− mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13−/− mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13−/− mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.
Highlights
The Rpn13 protein, previously termed adhesion-regulating protein 1 or GP110, has been identified as a tumor-associated gene product [1,2,3,4,5,6], and more recently as an amplification target in ovarian cancer [7]
Inverse genomic PCR of DNA isolated from Uch37 embryonic stem cells revealed that the gene-trap vector had inserted in intron 1 downstream of the first coding exon of the mouse Uchl5 gene which encodes Uch37 (Figure 1A) and was confirmed by genomic PCR analysis (Figure 1B)
Inactivation of Rpn13 gene in KO mice was confirmed by genomic PCR (Figure 1B), expression analysis of the gene transcript (Figure 1C), and by immunohistochemical (IHC) staining with a monoclonal antibody (mAb) to the Rpn13 protein (Figure 1D)
Summary
The Rpn protein, previously termed adhesion-regulating protein 1 or GP110, has been identified as a tumor-associated gene product [1,2,3,4,5,6], and more recently as an amplification target in ovarian cancer [7]. Rpn was suggested to regulate cell adhesion as a membrane-associated protein [2], but further examination revealed that it is primarily a cytosolic protein associated with proteasomes [8]. Independent studies demonstrated direct interaction of Rpn with ubiquitin and the deubiquitinating enzyme Uch ( termed UchL5) [9,10], as well as with the proteasome subunit Rpn2/S1 [11,12]. Rpn is emerging as a potentially important docking/coupling protein with a regulatory function in recognition and disassembly of ubiquitinated proteins at the proteasome
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