Báo cáo khoa học: Ubiquitin C-terminal hydrolase-L1 (Uch-L1) correlates with gonadal transformation in the rice field eel

Ubiquitin C-terminal hydrolase-L1 (Uch-L1) Correlates with Gonadal Transformation in the Rice Field Eel

Tác giả: Jinhua Sun, Xuan Shang, Yihao Tian, Wei Zhao, Yan He, Ke Chen, Hanhua Cheng and Rongjia Zhou

Lĩnh vực: Sinh học phát triển, Di truyền học

Nội dung tài liệu: Nghiên cứu này khám phá vai trò của enzyme Ubiquitin C-terminal hydrolase-L1 (Uch-L1) trong quá trình biến đổi tuyến sinh dục ở cá lóc (rice field eel), một loài động vật có xương sống có khả năng đảo ngược giới tính tự nhiên. Sử dụng phân tích biểu hiện gen và protein, nghiên cứu xác định rằng Uch-L1 được biểu hiện chủ yếu ở tuyến sinh dục (tinh hoàn, buồng trứng, và tuyến sinh dục lưỡng tính) và não. Đặc biệt, mức độ biểu hiện của Uch-L1 tăng lên trong giai đoạn biến đổi giới tính, từ giai đoạn liên giới tính trở đi. Nghiên cứu cũng chỉ ra rằng Uch-L1 có thể tương tác với ubiquitin, gợi ý vai trò của nó trong việc điều hòa cân bằng ubiquitin và hệ thống ubiquitin-proteasome. Sự biến đổi cấu trúc của Uch-L1, bao gồm dimer hóa và oligomer hóa, cũng thay đổi trong quá trình biến đổi giới tính, với tỷ lệ dimer hóa/oligomer hóa giảm dần. Những phát hiện này cho thấy Uch-L1 đóng vai trò quan trọng không chỉ trong quá trình sinh sản mà còn trong quá trình biến đổi tuyến sinh dục ở cá lóc.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 10 September 2007, revised 11 November 2007, accepted 15 November 2007)
• doi:10.1111/j.1742-4658.2007.06194.x
• The ubiquitin-proteasome pathway is crucial for a variety of biological processes, including spermatogenesis. Ubiquitin C-terminal hydrolase-L1 (Uch-L1) is thought to associate with monoubiquitin to control ubiquitin levels.
• Here, we report the identification of Uch-L1 cDNA from the testis of the rice field eel, a natural sex reversal vertebrate, by using cDNA microarray analysis.
• Uch-L1 encodes a protein of 220 amino acids that shows high homology to Uch-L1 of vertebrates, especially fish species.
• Both mRNA and protein are mainly expressed in testis, ovotestis and ovary, as well as in the brain.
• Immunohistochemistry analysis revealed differential expression of Uch-L1 in three kinds of gonads.
• In the ovary, expression of Uch-L1 was observed mainly in the developing ovary and slightly in the mature ovary.
• In ovotestis during the intersex stage, Uch-L1 was expressed in the male gonad epithelium and degraded ovary.
• In testis, expression was observed in developing germ cells, including spermatogonia and spermatocytes.
• Furthermore, Uch-L1 was upregulated during gonadal transformation, especially from the beginning of the intersex stage onwards.
• Native-PAGE showed that Uch-L1 underwent dimerization and oligomerization in gonads, and that the relative level of dimerization/oligomerization decreased during gonadal transformation.
• Simultaneously, ubiquitin polypeptide expression was upregulated during this process.
• These results suggest that Uch-L1, via the ubiquitin-proteasome system, may play an important role not only in gametogenesis, but also in the gonadal transformation process in the rice field eel.
• Ubiquitin is a 76 amino acid polypeptide that, as its name implies, is ubiquitously distributed and highly conserved throughout eukaryotic organisms, and plays a crucial role in a wide variety of biological processes, through covalently attaching to lysine residues of target proteins [1].
• Several ubiquitin molecules, in the form of a multiubiquitin chain, are conjugated to a substrate.
• Multiubiquitination serves mainly to label the substrate for degradation via the 26S proteasome system [2].
• The cellular ubiquitin balance is controlled by release of ubiquitin from uniquitin-conjugated unwanted, misfolded, or damaged polypeptides within the cell by deubiquitinating enzymes, a family of proteases that hydrolyze ubiquitin molecules from polyubiquitin chains after their release from target proteins [3].
• Ubiquitin C-terminal hydrolase-L1 (Uch-L1) is one of many deubiquitinating enzymes, and is selectively and abundantly expressed in neuron cells, testis and ovary [4,5] to hydrolyze ubiquitin from ubiquitinated cellular proteins, thereby deciding protein fate and degradation, or regulation of biological processes via the ubiquitination signal pathway.
• In addition to its hydrolase activity, Uch-L1 has also been shown to have in vitro ligase activity, and this ligase activity is correlated with dimerization of the enzyme, which may
• Abbreviations
• Results
• Identification of the evolutionarily conserved Uch-L1
• To search for genes involved in sexual differentiation, we utilized the natural sex reversal trait of the rice field eel, a teleost fish, as a new model system for vertebrate sexual development [14,15].
• We differentially screened a swamp eel testis cDNA library on microarrays using probes from testis, ovotestis and ovary mRNA respectively.
• A cDNA that was found to be differentially expressed among testis, ovotestis and ovary showed the greatest sequence similarity to vertebrate Uch-L1, especially to the fish branch (Fig. 1B).
• Uch-L1 of the rice field eel and the Nile tilapia was most closely related among the 18 samples (10 species).
• The Uch-L1 gene sequence predicted a protein of 220 amino acids.
• Uch-L1 is composed of two lobes, one consisting of five a-helices (a1, a3, a4, a5, and a6), and the other consisting of two a-helices (a2 and a7) and six β-strands (β1-β6) (Fig. 1A).
• Between the two lobes is a cleft in which the putative catalytic triad of C87, H159 and D174 resides, as determined by sequence similarity with other known Uch proteins.
• The overall architecture of eel Uch-L1 closely resembles that of human ubiquitin C-terminal hydrolase-L1 (UCH-L1).
• Uch-L1 was upregulated during gonadal transformation of the rice field eel
• To analyze expression patterns of Uch-L1, we carried out RT-PCR using RNAs isolated from gonads and other tissues.
• Uch-L1 was dominantly expressed in testis and ovotestis, slightly in ovary and brain, but not in heart, liver, kidney, or spleen (Fig. 2A).
• Northern blotting analysis confirmed the expression pattern; a main band of 1.2 kb was observed (Fig. 2B).
• Furthermore, accurate quantitative analysis was carried out by real-time fluorescent quantitative RT-PCR, which showed that the expression level of Uch-L1 in ovotestis I increased ~10-fold as compared with that in ovary (Fig. 2C).
• The expression level of Uch-L1 did not change significantly from ovotestis II to testis.
• Moreover, to investigate protein expression, western blot analysis was carried out, which showed a band of 26 kDa and further confirmed the expression pattern of the gene at the protein level (Fig. 3).
• Cellular localization of Uch-L1 protein in germ cells
• Immunohistochemical analysis of gonadal sections revealed different localizations of Uch-L1 in three kinds of gonads.
• In the ovary, localization of Uch-L1 was observed mainly in developing ovary and slightly in mature ovary (Fig. 4A).
• In ovotestis during the intersex stage, Uch-L1 was expressed in the male gonad epithelium and degraded ovary (Fig. 4D).
• In testis, expression signals were observed in developing germ cells, including spermatogonia, and spermatocytes (Fig. 4G).
• Dimerization and oligomerization of Uch-L1 in gonads
• Native-PAGE was used to determine whether Uch-L1 forms dimers or oligomers in gonads.
• When equal amounts of Uch-L1 from three kinds of gonads were sampled as a control (SDS/PAGE; Fig. 5B), native-PAGE analysis showed that dimers and oligomers of Uch-L1, as well as monomers, were observed in the three kinds of gonads.
• However, rates of dimerization and oligomerization were high in ovary and lower in ovotestis, whereas monomers were mainly formed in testis, which suggested that the dimerization/oligomerization level of Uch-L1 decreased during gonadal transformation from female to male (Fig. 5A).
• Ubiquitination was upregulated during gonadal transformation
• The main role of ubiquitin is as a targeting signal for degradation of ubiquitinated proteins.
• Coimmunoprecipitation was used to test the interaction between Uch-L1 and ubiquitin, and showed that Uch-L1 could bind to and interact with monoubiqutin in the rice field eel (Fig. 6).
• To test whether ubiquitination was upregulated during gonadal transformation
• polyubiquitin expression were relatively low in ovary, whereas during gonadal transformation, expression of both monoubiquitin and polyubiquitin was upregulated in ovotestis and maintained at a high level in testis (Fig. 7).
• Discussion
• M. albus taxonomically belongs to the teleosts, the family Synbranchidae of the order Synbranchiformes (Neoteleostei, Teleostei, Vertebrata). This freshwater fish is not only an economically important species in southeast Asia for food production, but also a good model for comparative genomic studies of distantly related vertebrate processes, and sexual differentiation, because of its primitive evolutionary status, relatively small genome size, and natural sex reversal via intersex from female to male during its life cycle [13,14].
• Taking advantage of the sex reversal characteristic of the rice field eel, we identified Uch-L1 cDNA by testis cDNA microarray analysis, which showed high homology to Uch-L1 of vertebrates, especially fish species.
• Uch-L1 is a member of the Uch family, members of which release free ubiquitin from polyubiquitin-conjugated proteins and conjugate ubiquitin to target proteins, because of their ligase activity [6].
• Therefore, Uch-L1 plays an important role in the maintenance of intracellular monoubiquitin levels by generating ubiquitin through the hydrolysis of polyubiquitinated proteins, and regulates the ubiquitin pathway.
• The main role of ubiquitin is as a targeting signal for degradation of ubiquitinated proteins.
• The ubiquitin pathway is responsible for the proteasome-mediated turnover of short-lived regulatory proteins as well as for the degradation of misfolded, unassembled or damaged proteins that could be potentially dangerous for the cell [1,2].
• The biological roles of Uch-L1 associated with ubiquitin in gonadal transformation of the rice field eel were further revealed in the present study.
• Our main findings are as follows: (a) Uch-L1 is upregulated during gonadal transformation of the rice field eel; (b) dimerization and oligomerization of Uch-L1 takes place in testis, ovary and ovotestis, and the dimerization/oligomerization level of Uch-L1 decreased during gonadal transformation from female to male; and (c) consistent with upregulation of Uch-L1, ubiquitination is upregulated during gonadal transformation, and Uch-L1 may interact first with ubiquitin, and thus join in maintaining the total ubiquitination level during gonadal transformation.
• These results suggest an important role of Uch-L1 associated with ubiquitin in gonadal transformation from female to male via intersex.
• Our studies showed that the expression of Uch-L1 in gonads of the rice field eel starts to increase during sex transition and remains at a high level in the mature testis.
• Native-PAGE analysis indicated that there are both hydrolase activity (monomer) and ligase activity (dimers-oligomers) of Uch-L1 in gonads.
• The enzyme activity of Uch-L1 was mostly that of a hydrolase, because the majority of the protein is in the monomeric form in all three kinds of gonads.
• Interestingly, the ratio of oligomerization (including dimerization) to monomerization decreased during gonadal transformation, which showed that ligase activity seems to be more important in oogenesis, whereas Uch-L1 monomer and upregulated ubiquitin may play key roles in gonadal transformation from ovotestis to testis.
• Uch-L1 can bind to and stabilize monoubiquitin; this is a function of Uch-L1 affinity for ubiquitin rather than its deubiquitinating activity [4].
• This interaction between Uch-L1 and ubiquitin may, at least partly, play an important role in maintaining the intracellular levels of ubiquitin during gonadal transformation.
• Total ubiquitination during gonadal transformation seems to be a complex process, and other unknown factors may also play a role in maintaining the total level of ubiquitination during gonadal transformation.
• During the course of sexual transformation, ovary was gradually degraded and formed ovotestis, and male epithelium rapidly developed and was finally replaced by testis.
• Therefore, many proteins were degraded during the ovotestis stage; these proteins were connected with the ubiquitin 26S proteasome degradation pathway.
• The expression level of Uch-L1 together with ubiquitin in ovotestis was highly upregulated, which is essential for the start of gonadal transformation.
• Uch-L1 may be also involved in germ cell apoptosis in the rice field eel during spermatogenesis, as observed in the mouse [7].
• These results suggested that Uch-L1, via the ubiquitin-proteasome system, may play an important role not only in gametogenesis, but also in the gonadal transformation process through macroregulation of hydrolase activity and microregulation of dimerization-dependent ligase activity, in the rice field eel.
• Experimental procedures
• Animals and antibodies
• The rice field eels were obtained from markets in the Wuhan area in China.
• Their sex was confirmed by microscopic analysis of gonad sections.
• The experiments were carried out in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.
• Antibody to Uch-L1 was supplied by K. Mochida.
• ẞ-Actin (sc-47778) was purchased from Santa Cruz Company (Santa Cruz, CA, USA).
• Antibody to polyubiquitin (D058-3) was purchased from MBL (Woburn, Japan), and antibody to monoubiquitin was supplied by S. Yokota.
• The secondary antibodies conjugated with alkaline phosphatase were purchased from Pierce Company (Rockford, IL, USA).
• Testis cDNA library construction and differential screening
• A testis cDNA library of the rice field eel was constructed using the SMART cDNA library construction kit, following the manufacturer’s protocol (Clontech, Mountain View, CA, USA).
• About 10 000 clones with insert sequences longer than 500 bp were dotted onto a Hybond N+ nylon membrane (Amersham, Chalfont St Giles, UK) by Qpix microarray (Genetix, New Milton, UK).
• Three micrograms each of mRNA from ovary, ovotestis and testis were reverse transcribed into respective cDNAs and labeled with [32P]dCTP[P].
• Three sets of arrays were hybridized overnight at 68 °C with the respective cDNA probe.
• Differentially expressed clones were sequenced.
• Uch-L1 structure prediction and phylogenetic analysis
• A homology model of Uch-L1 based on UCH-L1 [16] was created by the server SWISS-MODEL and viewed with SPDBV version 3.7.
• The structure of the rice field eel Uch-L1 was determined by the molecular replacement technique, using a homology model of Uch-L1 created from the atomic coordinates of UCH-L1 as a search model.
• The putative rice field eel Uch-L1 amino acid sequence from the nucleotide sequence of Uch-L1 was analyzed using the NCBI BlastP server.
• The Uch protein sequences from 10 species were aligned by CLUSTAL_X 1.8 [17].
• The neighbor-joining method was used to construct phylogenetic trees with PHYLIP (Phylogeny Inference Package, version 3.6).
• Bootstrap analyses with 2000 replicates for the neighbor-joining method were performed.
• RT-PCR
• Total RNAs were isolated using Trizol, and used as templates for reverse transcription using poly(T)18 primer and reverse transcriptase (Promega, Madison, WI, USA).
• RT-PCR was used to amplify Uch-L1.
• PCR amplification conditions were: 25 cycles of 30 s at 94 °C, 30 s at 64 °C and 30 s at 72 °C for Uch-L1; and 22 cycles of 25 s at 94 °C, 25 s at 64 °C and 25 s at 72 °C for Hprt.
• Primers were as follows: Uch-F, 5′-TTGGCGTGGGTGAAGTTGG-3′, and Uch-R 5′-CCTCACGGATTGCCTGGTTC-3′, for Uch-L1; Hprt-F, 5′-GA