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Identification and function of the testis proteasome

Author: DuBoYu
Tutor: ShenYan；QiuXiaoBo；WangLinFang
School: Peking Union Medical College , China
Course: Biochemistry and Molecular Biology
Keywords: Proteasome Testicular tissue Expression levels Spermatocytes Ubiquitination Synchronization Beijing Union Medical College Tissue sources Spleen tissue Cell differentiation chymotrypsin Spermatogenesis proteasome Polyubiquitin Subunit Spermatogenic cells Carrier protein Proteasome spermatocyte Ubiquitin ligase
CLC: Q55
Type: PhD thesis
Year: 2008
Downloads: 63
Quote: 0
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Abstract

The proteasome in the eukaryotic cells is composed of two parts,20S catalytic complex, which cleaves protein substrates, and the 19S regulatory complex, which binds one or both ends of 20S complex and regulates the activity of the 20S particle. The 20S catalytic complex is a symmetrical, cylinder-shaped particle composed of four rings (αββα). Three catalytic subunits PSMB6, PSMB7 and PSMB5 with chymotrypsin-like, trypsin-like and caspase-like activities lie within the two inside packedβrings. Under the induction of IFN-γ, these three catalytic subunits could be replaced by PSMB9, PSMB10 and PSMB8, which are a part of the immunoproteasome involved in antigen presentation.Previous results from our group have shown that two types of proteasomes different from the 26S proteasome can be purified from rabbit testes. These proteasomes contain PSME4 (a proteasomal activator with unknown functions), a new PSMA7-like subunit, and a few subunits usually present in the immunoproteasome. In the process of purification of the bovine muscle, testis and spleen proteasomes, we found that different concentrations of NaCl were needed to elute these proteasomes from an anion exchange column. By using the peptide fluorescence substrate, we determined the activities of the proteasome from these tissues and found that the chymotrypsin-like activity of testis proteasome was significantly lower than that from muscle and spleen (P＜0.05). The trypsin-like activity of the testis proteasome was significantly higher than that of the proteasome purified from muscle and spleen (P＜0.05). But the caspase like activity of muscle proteasome was significantly higher than that of the proteaosmes purified form testis and spleen (P＜0.05). No significant differences were observed when the two protein substrates histone H1 and casein were used to determine the degradation efficiency of these three kinds of proteasome (P>0.05). When several proteasome inhibitors (MG132, Velcade, Leupeptin and Ac-ApnLD-Al) were used to incubate with the proteasomes, no significant differences in the inhibition of the activities were observed among the proteasomes from testis, muscle and spleen (P>0.05).By using the real-time PCR and western blot experiments, we found similar expression levels of PSMB5 in the spermatogonia cell line(GC-1 spg), spermatocyte cell line(GC-2 spd) and somatic cell lines (NIH 3T3 and C2C12). But PSMB8 was found highly expressed in the spermatogonia cell line and the spermatocyte cell line and not detectable in the two somatic cell lines. We further found that the catalytic subunits PSMB8, PSMB9, PSMB6 and PSMB7, but not PSMB5 and PSMB10, could co-precipitate with PSME4. However, when the similar immunoprecipitation experiment was performed in NIH 3T3 cell lysates, no catalytic subunits were co-precipitated with PSME4 antibody, raising the possibility that PSME4 mostly does not associate with the 20S proteasome in somatic cells.Using the in-situ hybridization teniques, we also observed the expression pattern of PSME4, PSMB8 and PSMB5 in mouse testis. The expression patterns of the three selected genes were quite different in different stages of spermatogenesis. Prior to theⅧstages of mouse spermatogenesis, PSME4, PSMB8 and PSMB5 were observed in the round spermatids, but not in pachytene spermatocytes. After theⅧstages, the expression of PSME4, PSMB8 and PSMB5 was observed in pachytene spermatocytes, but not in spermatids, leptotene spermatocytes and sertoli cells. Moreover, according to the initial results of immunohistochemistry with anti-PSMB8 and anti-PSMB5, we observed high expression level of PSMB5 in the leydig cells, while PSMB8 was extensively expressed in the testis tissues. The above results suggested that in the germ cells of different developmental stages, the different compostions of proteasome might exist.In eukaryotic cells, the protein substrate of the 26S proteasome usually carries an ubiquitin tag, which is added through ubiquitination. The enzymes involved in the ubiquitination include ubiquitin-activating enzyme (E1), ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3). E1 activates the ubiquitin by promoting the formation of high-energy sulfur-ester bond in the presence of ATP. Then the activated ubiquitin molecule is transferred to an E2. At last the ubiquitin is transferred to the substrate by E3. After that, the substrate tagged with polyubiquitin chain is targeted for degradation by the 26S proteasome. Certain apoptotic proteins played important roles during sperm development. For example, the giant Inhibitor of Apoptosis Protein (IAP) BRUCE/Apollon, which acts as both E2 and E3, can protect the sperm nucleus from high dense condensation and degeneration in Droshophila, indicating that this protein might play a very important role during spermatogenesis. Recent studies showed that BRUCE controls the midbody ring formation at the final stages of mitotic cell cycle. As BRUCE was the substrate of the E3 ubiquitin ligase, Nrdp1/RNF41, we examined the protein levels of Nrdp1/RNF41 and BRUCE throughout the cell cycle progression. The protein levels of Nrdp1/RNF41 increased in the early G2/M phase (3 hours after the release of synchronization), reached its peak at 18-24 hours after the release. The change of BRUCE protein levels appeared a little behind that of Nrdpl. Its levels increased at 6 hours after the release and kept increasing afterwards. During the early stages of the release of synchronization (S stage), the lowest BRUCE protein levels were observed. Our results demonstrate that Nrdp1 might be involved in the regulation of the cell cycle by promoting ubiquitination and degradation of BRUCE. However, more experiments were still needed to address the roles of these proteins in the progression of male meiotic cell cycle during spermatogenesis.Taken together, our results suggest that there are specialized forms of proteasome in mammalian testis with distinct compositions and activity. In addition, we found that the ubiquitin ligase Nrdpl/RNF41 was probably involved in cell cycle regulation.

Identification and function of the testis proteasome

Abstract

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