中文责编:晨 兮; 英文责编:艾 琳
1)天津工业技术大学生物技术学院, 天津 300457; 2)中国科学院天津工业生物技术研究所,国家工业酶重点实验室,天津300308; 3)天津药物研究院天津分子设计与药物发现重点实验室, 天津300193; 4)深圳大学医学部,呼吸疾病国家重点实验室深圳大学变态反应分室, 深圳 518060
Su Wencheng1,2, Lyu Cao1,2, Shi Lili3, Jing Xiaofei1,2, Gai Yuanming2, Zhang Jie2, Tan Huanbo2, Wang Pengju2, Xia Lixin4, Zou Peijian2, and Qin Gang21)College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, P.R.China2)National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P.R.China3)Tianjin Key Laboratory of Molecular Design and Drug Discovery, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, P.R.China4)Health Science Center, Shenzhen University, State Key Laboratory of Respiratory Disease for Allergy at Shenzhen University, Shenzhen 518060, P.R.China
Ubiquitination modification is a dynamic process essential for eukaryotic cell physiology. Ubp3, the Saccharomyces cerevisiae homologue of human deubiquitinase USP10, together with its cofactor Bre5, plays an active role in numerous cellular processes. Although Bre5 is essential for Ubp3 function in vivo, unfortunately, due to difficulty in preparing critical quantities of intact functional Ubp3 and Ubp3/Bre5 reconstitute, systemic characterization on this complex is lacking. Hence, how exactly Bre5 regulates Ubp3 activity still remains elusive. To fill this gap, we report the successful expression and purification of recombinant Ubp3 and Bre5 in Escherichia coli in monomeric and complex form. To our knowledge, this is the first report the successful preparation of full-length Ubp3/Bre5 protein complex in large scale, which allows us to obtain further understanding of molecular bases. The stoichiometric interaction between purified Ubp3 and Bre5 confirmed proper folding of these proteins. To assess the proposed direct interactions between Ubp3 and Bre5 with the ubiquitin selective ATPase associated with a variety of cellular activities(AAA ATPase)Cdc48, series of pull-down assays are performed; results reveal that, neither Ubp3 nor Bre5 alone is able to bind Cdc48. However, the Ubp3/Bre5 complex could bind Cdc48 efficiently, which provids novel insight on Ubp3/Bre5-Cdc48 interaction mode. In summary, our results lay the foundation for future mechanistic evaluation by both biochemical and structural means.
The dynamic balance between ubiquitination and deubiquitination, the two reversal post-translational regulation processes, plays a vital role in eukaryotic cell physiology facilitated by highly specific catalytic machinery. Deubiquitination is catalyzed by deubiquitinating proteases(DUBs), which are mainly categorized into five groups based on structural homology: ubiquitin-specific processing proteases(USPs/UBPs), ubiquitin C-terminal hydrolases(UCHs), ovarian tumor domain-containing proteases(OTUs), Ataxin-3-like proteases and Jab1/Mov34/Mpr1 Pad1 N-terminal+(MPN+)(JAmmol/L)proteases[2-3]. Among DUBs, UBPs represent the most abundant type. In the Saccharomyces cerevisiae genome, at least 16 UBP encoding sequences have been discovered. None of them is essential for cell viability, but certain individual mutants exhibit pleiotropic abnormalities, implicating important and widespread roles in cellular functions[4-5].
Ubp3, the Saccharomyces cerevisiae homologue of human USP10, has been shown to be involved in regulating multiple cellular processes, including DNA repair[6-7], transcription regulation[8-9], signal transduction[10-11], anterograde/retrograde transport and ribophagy. One positive regulator named Bre5 directly interacts with Ubp3 and is indispensable for Ubp3 function. Initial structural characterization has revealed that Ubp3 and Bre5 form a symmetric heterotetramer in which the Bre5 NTF2-like domain dimer interacts with two N-terminal motifs of Ubp3 with apparent 1:1 stoichiometry[14-15]. The effect of cofactor Bre5 on Ubp3 is to either facilitate substrate targeting, or to modulate its catalytic activity or to achieve both; unfortunately, direct proof from biochemical and structural aspects is still deficient due to difficulty in preparing the large Ubp3 and full-length functional Ubp3/Bre5 reconstitute. Recently, one intriguing connection of the ubiquitin-selective chaperon Cdc48, and its cofactor Ufd3 to Ubp3/Bre5 mediated ribophagy was proposed. Ubp3 and Bre5 were shown to interact with Cdc48 and Ufd3 directly. However, the molecular basis on these interactions and functional mechanism underlying them remain to be precisely evaluated.
Prior attempts to purify recombinant full-length Ubp3 were not very successful. We find that this was mostly due to the intrinsic instability of N-terminal region of Ubp3(our unpublished data). To initiate systemic characterization of the deubiquitinating complex Ubp3/Bre5, we report successful expression and purification of recombinant Ubp3 and Bre5 in a monomeric and complex form, through the Escherichia coli(E. coli)expression system. Utilizing these purified samples, we carefully assess the interactions between Cdc48 and Ubp3/Bre5 in vitro. As far as we know, this is the first report on the successful preparation of a full-length Ubp3/Bre5 complex in large scale, which allows us to obtain further understanding of molecular basis of the Ubp3/Bre5 via biochemical and structural biology means.
E. coli DH5α, E. coli BL21(DE3), E. coli trx(DE3)and T4 DNA Ligase were purchased from Beijing TransGen Biotech(Beijing, China). pGEX- 4T-1, pET-28a and pET-32-Bre5 were obtained from our laboratory. Restriction enzymes were purchased from Fermentas Life Sciences(Vilnius, Lithuania). Es Taq DNA Polymerases were purchased from Beijing CoWin Bioscience Co., Ltd(Beijing, China). Extraction Kit, Plasmid Mini Kit and Cycle-pure Kit were purchased from OMEGA Bio-Tek(Norcross,GA). Primers were ordered from Shanghai Sangon Biotechnology(Shanghai, China). IPTG was purchased from Sigma(CA, USA). Recombinant glutathione S-transferase(GST), GST-Cdc48 and 6×His-Cdc48 were previously prepared as reported.
The Ubp3 encoding sequence was amplified from Saccharomyces cerevisiae genome, with primers designed with restriction endonuclease cloning sites EcoR I and Xho I(Table 1). The PCR reactions were carried out as: Step 1 94 ℃,2 min; Step 2 94 ℃,30 s, 55 ℃,30 s, 72 ℃,2 min; Step 3 72 ℃, 10 min, with 25 cycles of step 2. The recovered PCR product and vector pGEX- 4T-1 were digested with EcoR I and Xho I restriction enzymes and were ligated via T4 ligase, then transformed into E. coli DH5α cells. Positive clones designated as pGEX- 4T-1-Ubp3 were selected by colony PCR and were verified by DNA sequencing.
DNA fragments encoding Bre5 gene was amplified by PCR from a previously constructed pET-32-Bre5 plasmid with primers designed with restriction endonuclease cloning sites Nco I and Xho I(Table 1). PCR reactions were carried out as: Step 1 95 ℃, 2 min; Step 2 94 ℃, 30 s, 55 ℃, 30 s, 72 ℃, 1 min 30 s; Step 3 72 ℃, 10 min, with 25 cycles of step 2. The amplified Bre5 fragment was cloned into a pET-28a vector by the similar process described above. Positive clones were selected via double digestion, and the sequencing verified plasmid was designated as pET-28a-Bre5.
pGEX- 4T-1-Ubp3 and pET-28a-Bre5 plasmids were transformed into E. coli trx(DE3)and E. coli BL21(DE3)cells respectively. A single colony was inoculated into a LB medium supplemented with proper antibiotics(100 μg/mL ampicillin or 50 μg/mL kanamycin)and cultivated overnight at 37 ℃ with vigorous shaking. The overnight cultures were diluted in fresh pre-warmed medium(including proper antibiotics)and grown at 37 ℃ with vigorous shaking, until the OD600 reached 0.5- 0.7. Protein expression was induced by adding IPTG(final concentration at 0.2 mmol/L for GST-Ubp3 induction and 0.1 mmol/L for 6×His-Bre5 induction), and cultures were collected after overnight growth at either 25 ℃ or 16 ℃. Samples were sonicated and fractionated, and whole cell lysate, supernatant, and pellet fractions were analyzed by means of sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE)and Coomassie staining.
The co-expression experiment was essentially described above, except that both pGEX- 4T-1-Ubp3 and pET-28a-Bre5 were co-transformed into E. coli BL21(DE3)in the presence of antibiotics(100 μg/mL ampicillin plus 50 μg/mL kanamycin).
To purify recombinant GST-Ubp3 in large scale, cultivated cells with optimal induction were harvested by centrifugation(4 500 r/min, 20 min, 4 ℃)and re-suspended in ice cold buffer(PBS, pH=7.4, 0.4 mmol/L of PMSF, 1× protease inhibitor, 3 mmol/L DTT)and lysed via French press. The lysate was then centrifuged at 15 000 r/min for 30 min at 4 ℃, with the supernatant applied to Glutathione Sepharose resin(GE Healthcare cat. #17-5132- 03)pre-equilibrated with PBS and incubated with rotating for 4 h at 4 ℃. The column was washed with equilibration buffer(PBS containing 3 mmol/L DTT, 0.1% Triton X-100), and the protein was eluted with elution buffer(50 mmol/L Tris-HCl, pH=8.0, 200 mmol/L NaCl, 10 mmol/L glutathione). The elution fractions were analyzed via SDS-PAGE and Coomassie staining. Selected elution fractions were combined and dialyzed against ice-cold dialysis buffer(50 mmol/L Tris-HCl, pH=7.5, 50 mmol/L NaCl, 5% glycerol), then applied to the SP Sepharose FF(GE Healthcare cat. #17- 0929-01)pre-equilibrated with an ice-cold dialysis buffer. After being washed with 10 bed volumes of the same buffer, the column was eluted with an elution buffer containing a stepwise increase in salt concentration(0.2, 0.3 and 0.4 mol/L NaCl, respectively). The elution fractions were analyzed via SDS-PAGE and Coomassie staining. The concentration of purified recombinant GST-Ubp3 protein was determined by using bovine serum albumin as a standard.
To purify recombinant 6×His-Bre5 on a in large scale, cells were harvested by centrifugation(4 500 r/min, 20 min, 4 ℃)and pellets were re-suspended in 100 mL of lysis buffer(50 mmol/L Tris-HCl, pH=7.5, 150 mmol/L NaCl and 20 mmol/L imidazole, 0.4 mmol/L of PMSF, 5 mmol/L β-mercaptoethanol). Cells were lysed via French Press. Lysates were clarified(15 000 r/min, 30 min, 4 ℃), and the supernatants were transferred to Ni Sepharose FF(GE Healthcare cat. #17-5318-03)pre-equilibrated with lysis buffer and rotated for 2 h at 4 ℃. The column was sequentially washed with a wash buffer(50 mmol/L Tris, pH=7.5, 150 mmol/L NaCl, 0.1% Triton X-100, 5 mmol/L β-mercaptoethanol)containing a stepwise increase of imidazole concentrations(20, 50 and 100 mmol/L). Then the 6×His-Bre5 protein was eluted with elution buffer(50 mmol/L Tris, pH=7.5, 150 mmol/L NaCl, 250 mmol/L imidazole, 5 mmol/L β-mercaptoethanol), and elution fractions were analyzed via SDS-PAGE and Coomassie staining. The concentration of purified recombinant 6×His-Bre5 protein was determined by using bovine serum albumin as a standard.
For the large-scale Ubp3/Bre5 complex purification, GST-Ubp3 was purified as described above, except that after GST-Ubp3 binding, sufficient amount of purified 6×His-Bre5 was applied to glutathione column and incubated for 1 h at 4 ℃.
For protein identification LC-MS/MS analysis was conducted using LTQ XL from Thermo Fisher(ESI-MS/MS). The instrument was operated with a spray voltage of 3.5 kV and an ion transfer tube temperature of 25 ℃. The information-dependent acquisition(IDA)mode of operation was employed in which a survey scan from m/z 400 to 1 800 was acquired followed by collision-induced dissociation(CID), and for MS/MS, using a normalized collision energy of 35% with an activation q of 0.25 for 30 ms. Ion selection thresholds for MS and MS/MS were 1 000 and 500 counts, respectively.
Tandem mass spectra were extracted by the Xcalibur version 22.214.171.124. All MS/MS samples were analyzed using Sequest. Iodoacetamide derivative of Cys, de-amidation of Asn and Gln, oxidation of Met were specified in Sequest as variable modifications. Proteome Discoverer 1.2 was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at probability greater than 95.0% as specified by the result filter, which is Xcorr> 1.9 if the charge is 1, Xcorr> 2.2 if the charge is 2, Xcorr> 3.75 if the charge is 3. Protein identifications were accepted if they were established at probability greater than 99.0% and contained at least 2 identified unique peptides.
For pull-down assays, GST or GST fusion proteins were first incubated with 50 μL of pre-equilibrated glutathione-Sepharose beads in buffer A(50 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L DTT, 0.1% triton X-100, pH=7.5)for 1 h at 4 ℃. The beads were washed once with 500 μL of buffer A to remove unbound material and then incubated with prey proteins for 1 h at 4 ℃. Beads were washed three times with 1 mL of buffer A, followed by three times of wash with buffer B(50 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L DTT, pH=7.5), then mixed with an SDS-PAGE loading buffer and analyzed on SDS-PAGE.
Due to the exceptional ability of GST tag to greatly enhance the solubility and stability of fused proteins, GST tag has been widely used for facilitating recombinant protein preparation; therefore we introduce a GST domain fused at the N-term of Ubp3. The Upb3 gene was amplified using Saccharomyces cerevisiae genome DNA as a template, a single band at about 2.8 kb was obtained, in accordance with the size of Upb3 coding region(Fig.1(a)); the encoding fragment was inserted into bacterial expression vector pGEX- 4T-1, and a positive plasmid designated as pGEX- 4T-1-Ubp3 was selected via colony PCR(Fig.1(b))and verified via DNA sequencing. The Bre 5 gene was amplified similarly, with a fragment of about 1.5 kb obtained(Fig.1(c)), the encoding fragment was inserted into pET-28a to introduce a 6×His tag at N-term of Bre5. The positive plasmid, designated pET-28a-Bre5, was selected by double restriction enzyme digestion(Fig.1(d))and confirmed via DNA sequencing.
(a)PCR amplification of Ubp3 coding region from Saccharomyces cerevisiae genome. Lane 1, DNA marker; Lane 2, PCR product.(b)Verification of expression plasmids pGEX- 4T-1-Ubp3 by colony PCR. Lane 1, DNA marker; Lane 2-3, PCR amplified fragments verifying two positive clones.(c)PCR amplification of Bre5 coding region from a previously constructed plasmid pET-32-Bre5. Lane 1, DNA marker; Lane 2, PCR product.(d)Verification of expression plasmids pET-28a-Bre5 by restriction enzyme digestion. Lane 1, DNA marker; Lane 2-3, two positive clones digested with Nco I and Xho I.
Small scale expression trials of Ubp3 in E. coli trx(DE3)were performed at various induction conditions. Compared to non-induced condition, one band migrating at about 130 kDa became apparent after Isopropyl β-D-1-thiogalactopyranoside(IPTG)induction with 0.2 mmol/L IPTG at 16 ℃, which corresponds to the GST-Ubp3 fusion, thus resulted in a relatively higher solubility of the induced protein(Fig.2(a)); therefore, we chose the same induction condition for large scale preparation. As shown in Fig.2(b), after a single-step glutathione column purification, GST-Ubp3 was enriched in elution fractions. However, the same fractions also contained several contaminating components of diverse molecular weight. The size of a prominent contaminant was about 26 kDa(Fig.2(b), Lane 6), similar to an intact GST domain, which is not surprising since GST truncates are frequently co-purified with GST fusion proteins, especially when the fused partner contains degradation-prone areas. Other major contaminating proteins appeared to be the degradation intermediates of Ubp3, since these bands remained rather unstable, almost disappeared during dialysis(compared Fig.2(b), Lane 6 with Fig.2(c), Lane 1). To further improve the purity of combined GST-Ubp3 pool and especially to remove GST truncates, we continued with ion-exchange chromatography. Based on the estimated pIs for Ubp3 and GST(7.9 for Ubp3 versus 4.5 for GST), SP sepharose was selected, and the efficacy of contaminant removal was shown in Fig.2(c). GST truncates in dialysis buffer remained poorly bound to SP resin clearly hence largely existed in flowthrough(Fig.2(c), Lane 2); in contrast, most GST-Ubp3 adsorbed to SP resin at the same condition, and was able to be efficiently eluted when NaCl concentration was increased to 0.2- 0.3 mol/L(Fig.2(c), Lane 6-9). Recovered full-length GST-Ubp3 exhibited significant improvement on purity(>85%), its identity was uniquely verified by LC-MS/MS(Fig.3). Total yields of recombinant GST-Ubp3 are listed in Table 2.
(a)Small scale expression trials of GST-Ubp3 induced at 16 ℃(Lane 1- 4)and 25 ℃(Lane 5-8)respectively. Lane 1 and 5, total proteins of uninduced cells; Lane 2 and 6, total proteins of induced cells; Lane 3 and 7, soluble fraction of induced cells; Lane 4 and 8, insoluble fraction of induced cells.(b)GST-Ubp3 purification through glutathione column chromatography. Lane 1, soluble fraction of induced cells; Lane 2, flowthrough; Lane 3- 4, wash; Lane 5-8, elution fraction.(c)Further purification of GST-Ubp3 through SP cation-exchange chromatography. Lane 1, dialyzed GST-Ubp3 pool from glutathione column chromatography; Lane 2, flowthrough; Lane 3, wash; Lane 4-6, 0.2 mol/L NaCl elution fraction; Lane 7-9, 0.3 mol/L NaCl elution fraction; Lane 10, 0.4 mol/L NaCl elution fraction.
Amino acid sequence corresponding to Ubp3 was shown, with identified unique peptides highlighted in gray.
In experiments parallel to Ubp3, expression trials of Bre5 in E.coli BL21(DE3)were also performed. Upon induction, one protein with a molecular weight of about 70 kDa appears(Fig.4(a)), which is larger than the expected size of 6×His-Bre5(about 58 kDa); this is most likely due to unusual mobility of Bre5 in SDS-PAGE, since the identity of purified protein was confidently verified as Bre5 by LC-MS/MS(Fig.5). Target protein induced with 0.1 mmol/L IPTG at 16 ℃ exhibited relatively better solubility(Fig.4(a)), same induction condition was also applied to large scale purification. 6×His-Bre5 was prepared according to standard one-step nickel affinity chromatography procedure, as shown in Fig.4(b). The imidazole elution fractions were pooled and dialyzed, total yields of recombinant 6×His-Bre5 are summarized in Table 2.
(a)Small scale expression trials of 6×His-Bre5 induced at 16 ℃(Lane 1- 4)and 25 ℃(Lane 5-8)respectively. Lane 1 and 5, total proteins of uninduced cells; Lane 2 and 6, total proteins of induced cells; Lane 3 and 7, soluble fraction of induced cells; Lane 4 and 8, insoluble fraction of induced cells.(b)6×His-Bre5 purification through nickel affinity chromatography. Lane 1, soluble fraction of induced cells; Lane 2, flowthrough; Lane 3, wash; Lane 4, 50 mmol/L imidazole elution fraction; Lane 5- 6, 80 mmol/L imidazole elution fraction; Lane 7-9, 100 mmol/L imidazole elution fraction; Lane 10-12, 250 mmol/L imidazole elution fraction; Lane 13, 500 mmol/L imidazole elution fraction.
Amino acid sequence corresponding to Bre5 was shown, with identified unique peptides highlighted in gray.
Having successfully obtained soluble Ubp3 and Bre5 in high purity, we sought to confirm whether they are properly folded or not. Previously, it has been well established that Ubp3 and Bre5 physically interact with each other in vivo and in vitro[12, 14-15]; thus, we performed a GST-pulldown to directly examine the interaction. As shown in Fig.6, in contrast to GST(Lane 4), GST-Ubp3 displays a stoichiometric interaction with Bre5(Lane 5), consistent with previous reports[14-15]. Based on these data, we conclud that our prepared recombinant Ubp3 and Bre5 are functional.
Lane 1, GST; Lane 2, GST-Ubp3; Lane 3, 6×His-Bre5; Lane 4, pulldown sample using GST as bait and 6×His-Bre5 as prey; Lane 5, pulldown sample using GST-Ubp3 as bait and 6×His-Bre5 as prey.
The successful purification of functional Ubp3 and Bre5 individually prompted us to try preparing Ubp3/Bre5 complex directly, which is essential for further functional and structural characterization. Initially, we took our effort on co-expressing pGEX- 4T-1-Ubp3 and pET-28a-Bre5 in E.coli. Unfortunately the induction of Ubp3 and Bre5 is not at comparable level(Fig.7(a)), GST-Ubp3 induction is nearly undetectable), preventing productive complex assembly in vivo. To solve this problem, we developed a‘hybrid'procedure as shown in Fig.7(b), GST-Ubp3 was purified according to the established two-step procedure described above, except that after GST-Ubp3 binding to glutathione column, sufficient amount of recombinant 6×His-Bre5 was added to trigger the on-column complex assembly. By following this strategy, the glutathione elution fractions displayed nearly stoichiometric distributions of Ubp3 and Bre5, indicating successful complex formation on glutathione column(Fig.7(c)). Moreover, successive SP cation-exchange chromatography significantly enhanced the purity of Ubp3/Bre5 complex by efficiently removing GST truncates and other contaminants, a procedure comparable to individual Ubp3 purification(Fig.7(d)); interestingly, the preformed Ubp3/Bre5 complex well tolerated high salt elution condition(0.4- 0.5 mol/L NaCl), presenting exceptional stability.
(a)Small-scale co-expression trials of GST-Ubp3 and 6×His-Bre5 induced at 16 ℃(Lane 1- 4)and 25 ℃(Lane 5-8)respectively. Lane 1 and 5, total proteins of un-induced cells; Lane 2 and 6, total proteins of induced cells; Lane 3 and 7, soluble fraction of induced cells; Lane 4 and 8, insoluble fraction of induced cells.(b)Procedure for two-round GST-Ubp3/6×His-Bre5 complex preparation.(c)The first round of GST-Ubp3/6×His-Bre5 complex preparation through glutathione column chromatography. Lane 1, soluble fraction of induced cells; Lane 2, flowthrough after 6×His-Bre5 incubation with glutathione column; Lane 3-7, wash; Lane 8-11, elution fraction.(d)The second round of GST-Ubp3/6×His-Bre5 complex purification through SP cation-exchange chromatography. Lane 1, dialyzed GST-Ubp3/6×His-Bre5 pool from glutathione column chromatography; Lane 2, flowthrough; Lane 3- 4, wash; Lane 5, 0.2 mol/L NaCl elution fraction; Lane 6-7, 0.3 mol/L NaCl elution fraction; Lane 8-10, 0.4 mol/L NaCl elution fraction.
Recently, the Ubp3/Bre5 complex has been linked to the ATPase associated with a variety of cellular activities(AAA ATPase)Cdc48. Ossareh-Nazari et al proposed a direct interaction between Ubp3 and Bre5 with Cdc48 respectively, hence providing further evidence that Cdc48 has close crosstalk with deubiquitinating pathways. Taking advantages of Ubp3, Bre5 and Ubp3/Bre5 preparations, we directly examined the proposed interactions by performing a series of GST-pulldown experiments. To our surprise, we could hardly observe any interaction between GST-Ubp3 and Cdc48(Fig.8(a), Lane 5), consistently. GST-Cdc48 also failed to pulldown Bre5(Fig.8(b), Lane 3); these results are obviously contradictory to the former finding from Ossareh-Nazari et al. Intriguingly, however, when the preformed GST-Ubp3/Bre5 complex was used as pulldown bait, a significant binding of Cdc48 was observed(Fig.8(c), Lane 3), indicating the assembled Ubp3/Bre5 complex is indeed able to physically interact with Cdc48. At this moment, we could not explain the discrepancy, but our results strongly suggest that more careful experiments need to be performed to uncover the real Ubp3/Bre5-Cdc48 interaction mode.
(a)GST-Ubp3 fails to interact with 6×His-Cdc48. Lane 1, 6×His-Bre5; Lane 2, 6×His-Cdc48; Lane 3, pulldown sample using GST as bait and 6×His-Cdc48 as prey; Lane 4, pulldown sample using GST-Ubp3 as bait and 6×His-Bre5 as prey; Lane 5, pulldown sample using GST-Ubp3 as bait and 6×His-Cdc48 as prey.(b)GST-Cdc48 fails to interact with 6×His-Bre5. Lane 1, 6×His-Bre5; Lane 2, pulldown sample using GST as bait and 6×His-Bre5 as prey; Lane 3, pulldown sample using GST-Cdc48 as bait and 6×His-Bre5 as prey.(c)GST-Ubp3 /6×His-Bre5 complex interacts with 6×His-Cdc48. Lane 1, 6×His-Cdc48; Lane 2, pulldown sample using GST as bait and 6×His-Cdc48 as prey; Lane 3, pulldown sample using GST-Ubp3/6×His-Bre5 complex as bait and 6×His-Cdc48 as prey.
The pleiotropic defects of ubp3 mutant indicate its widespread cellular functions. Despite the involvement of deubiquitinating activity of Ubp3/Bre5 in multiple cellular processes and partial resolution of Ubp3/Bre5 interaction mode, the molecular basis on how Bre5 regulates Ubp3 activity is still largely unknown. To precisely dissect the potential role of Bre5 in step of substrate recognition, catalytic activation or crosstalk with other interacting partners, a sophisticated in vitro reconstitution system is highly demanding. Obviously the successful preparation of functional Ubp3/Bre5 complex in high homogeneity is a prerequisite. In this work, we have established an expression and purification system to fulfill this requirement. With the development of a simple and efficient purification strategy we could obtain recombinant full-length Ubp3, Bre5 and also Ubp3/Bre5 complex in large scale, which to our knowledge has not been reported before.
Cdc48, a highly conserved component in AAA ATPase family, was noticed and studied recently, because of its distinct ubiquitin-selective property: the homohexamer of Cdc48 can act as a general platform for multi-purpose decision making of molecular events, depending on its ability to interact with plenty of cofactors, among which both ubiquitin ligases and DUBs are included[18-19]. The discovery of Ubp3-Cdc48 interaction further enriches the toolbox of Cdc48 and extends its action in ribophagy pathway. To confirm this important notion and obtain deeper insight, we set up a series of interaction assays to assess the proposed one-to-one interactions between Ubp3, Bre5 and Cdc48. Importantly, in our system we could not reproduce the discovered physical interaction between Ubp3 and Bre5 with Cdc48, however, we could detect a stable interaction between Ubp3/Bre5 complex and Cdc48, implicating synergistic action of Ubp3 and Bre5 upon Cdc48 binding. This perspective could potentially explain the obligatory role of Bre5 in Ubp3 functioning. Undoubtedly, revision of the current working model awaits more careful experiments.
We believe that the high purity reconstitutes of recombinant Ubp3/Bre5 will continuously bring deeper insight o molecular properties of this complex in future. Importantly, in an established in vitro enzymatic activity assay, the Ubp3/Bre5 complex can exhibit typical deubiquitinating enzyme activity(manuscript in preparation), which opens up apath for systemic catalytic mechanism characterization of Ubp3.