E3-independent ubiquitination of AtMAPR/MSBP1

AtMAPR5/MSBP1 and its homologs can be ubiquitinated in the absence of E3 ligase in in vitro ubiquitination assays. Ubiquitinated AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 were identified using LC–MS/MS. Analysis of trypsin-released signature peptides showed that this E3-independent ubiquitination of AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 was dominated by mono-ubiquitination at multiple sites. Unlike AtUBC8-type E2s, AtUBC36 was not able to transfer ubiquitin to AtMAPR2. The truncated mutants AtMAPR2Δ1–10, AtMAPR2Δ1–30, and AtMAPR2_1–73 could also be ubiquitinated. The presence of a ubiquitin-binding domain (UBD) allows proteins to be ubiquitinated independently of E3 ligases. However, AtMAPRs do not contain any known UBD. In vitro ubiquitination of AtMAPR2 observed in this study will be further studied in biochemical and physiological aspects.     

Ubiquitination without E3

In the current issue of Molecular Cell, Hoeller et al. (2007) demonstrated that proteins bearing ubiquitin-binding domains (UBDs) can be ubiquitinated directly by E2-conjugating enzymes, bypassing the requirement for E3 ubiquitin ligases.     

Autoubiquitination of BCA2 RING E3 ligase regulates its own stability and affects cell migration

Accumulating evidence suggests that ubiquitination plays a role in cancer by changing the function of key cellular proteins. Previously, we isolated BCA2 gene from a library enriched for breast tumor mRNAs. The BCA2 protein is a RING-type E3 ubiquitin ligase and is overexpressed in human breast tumors. In order to deduce the biochemical and biological function of BCA2, we searched for BCA2-binding partners using human breast and fetal brain cDNA libraries and BacterioMatch two-hybrid system. We identified 62 interacting partners, the majority of which were found to encode ubiquitin precursor proteins including ubiquitin C and ubiquitin A-52. Using several deletion and point mutants, we found that the BCA2 zinc finger (BZF) domain at the NH(2) terminus specifically binds ubiquitin and ubiquitinated proteins. The autoubiquitination activity of BCA2, RING-H2 mutant, BZF mutant, and various lysine mutants of BCA2 were investigated. Our results indicate that the BCA2 protein is strongly ubiquitinated and no ubiquitination is detected with the BCA2 RING-H2 mutant, indicating that the RING domain is essential for autoubiquitination. Mutation of the K26 and K32 lysines in the BZF domain also abrogated autoubiquitination activity. Interestingly, mutation of the K232 and K260 lysines in and near the RING domain resulted in an increase in autoubiquitination activity. Additionally, in cellular migration assays, BCA2 mutants showed altered cell motility compared with wild-type BCA2. On the basis of these findings, we propose that BCA2 might be an important factor regulating breast cancer cell migration/metastasis. We put forward a novel model for BCA2 E3 ligase-mediated cell regulation.     

UHRF2, another E3 ubiquitin ligase for p53

UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.     

HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21（DE3）感受态细胞,经IPTG诱导,再进行SDS－PAGE,可得到有一条约34kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带。该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示：目的蛋白占整个菌体蛋白的36％以上,经Ni-柱纯化的E2蛋白纯度可达95％以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阳性血清,结果表明15份抗HCV阳必血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体。     

A new scheme to discover functional associations and regulatory networks of E3 ubiquitin ligases

Background

Protein ubiquitination catalyzed by E3 ubiquitin ligases play important modulatory roles in various biological processes. With the emergence of high-throughput mass spectrometry technology, the proteomics research community embraced the development of numerous experimental methods for the determination of ubiquitination sites. The result is an accumulation of ubiquitinome data, coupled with a lack of available resources for investigating the regulatory networks among E3 ligases and ubiquitinated proteins. In this study, by integrating existing ubiquitinome data, experimentally validated E3 ligases and established protein-protein interactions, we have devised a strategy to construct a comprehensive map of protein ubiquitination networks.

Results

In total, 41,392 experimentally verified ubiquitination sites from 12,786 ubiquitinated proteins of humans have been obtained for this study. Additional 494 E3 ligases along with 1220 functional annotations and 28588 protein domains were manually curated. To characterize the regulatory networks among E3 ligases and ubiquitinated proteins, a well-established network viewer was utilized for the exploration of ubiquitination networks from 40892 protein-protein interactions. The effectiveness of the proposed approach was demonstrated in a case study examining E3 ligases involved in the ubiquitination of tumor suppressor p53. In addition to Mdm2, a known regulator of p53, the investigation also revealed other potential E3 ligases that may participate in the ubiquitination of p53.

Conclusion

Aside from the ability to facilitate comprehensive investigations of protein ubiquitination networks, by integrating information regarding protein-protein interactions and substrate specificities, the proposed method could discover potential E3 ligases for ubiquitinated proteins. Our strategy presents an efficient means for the preliminary screen of ubiquitination networks and overcomes the challenge as a result of limited knowledge about E3 ligase-regulated ubiquitination.

     

Ubiquitin conjugation to endogenous proteins in the dormant tuber of Helianthus tuberosus and during the first cell cycle

Ubiquitin is a small protein involved in an ATP-dependent proteolytic pathway in all eukaryotes. This pathway has been demonstrated to be required for both the bulk degradation of cellular proteins and the targeted proteolysis of specific regulatory proteins. We have investigated the presence of ubiquitin (Ub) and the ubiquitin-conjugating system in dormant and activated tubers of Helianthus tuberosus L. cv. OB 1 that represent a widely used model system for studies on the cell cycle in plants. Immunoblot experiments revealed the presence of free ubiquitin and ubiquitin conjugates. Furthermore, the presence of an active ubiquitin-conjugating system, both time- and ATP-dependent, was demonstrated by incubation with ¹²⁵I-labeled ubiquitin. A few proteins able to form thiol esters with ¹²⁵I-Ub and probably corresponding to ubiquitin-conjugating enzymes, E1 and E2s, were also found. During the first cell cycle, several proteins become ubiquitinated. In particular a large amount of protein conjugates was present at 6 h when the lowest content of free ubiquitin was found. Subsequently, a dramatic decrease in ubiquitin conjugates occurred. It is well known that cell cycle progression in eukaryotes depends on cyclin levels and cyclin B degradation is ubiquitin- and ATP-dependent. By immunoblot experiments we showed that cyclin B in H. tuberosus is present as at least two protein bands of 50 and 54 kDa and that their amounts undergo profound changes during the cell cycle. The 54-kDa band was also recognized by an anti-ubiquitin antibody. These data seem to indicate that in H. tuberosus activated tuber slices, the ATP-dependent ubiquitin proteolytic pathway is involved in the dedifferentiation process occurring after the artificial break of dormancy when the cells acquire the characteristics linked to the meristematic state.     

Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A

To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.     

Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

Background

In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.

Results

To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.

Conclusions

These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.     

BH3-ligand regulates access of MCL-1 to its E3 ligase

A genome wide search for new BH3-containing Bcl-2 family members was conducted using position weight matrices (PWM) and identified a large (480 kDa), novel BH3-only protein, originally called LASU1 (now also known as Ureb-1, E3^histone, ARF-BP1, and Mule). We demonstrated that LASU1 is an E3 ligase that ubiquitinated Mcl-1 in vitro and was required for its proteasome-dependent degradation in HeLa cells. Of note, the BH3 domain of LASU1 interacted with Mcl-1 but not with Bcl-2 or Bcl-Xl. A competing BH3-ligand derived from Bim interacted with Mcl-1 and prevented its interaction with LASU1 in HeLa cells, causing elevation of the steady-state levels of Mcl-1. This suggests that the unliganded form of Mcl-1 is sensitive to LASU1-mediated degradation of Mcl-1.     

重组人钙调素的制备及对细胞增殖作用影响的研究

The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was usd as template. After inserting the hCaM III cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA-recombinant expression vector(hCaM3/pBV220). The recombinant plasmid was then transformed into E. coli DH5 alpha. After heat induction, a high level expression of CaM protein was obtained. SDS-PAGE analysis showed that the recombinant E. coli could express a 17 kD protein which accounted for about 20% of the total cellular protein. Western blot analysis showed that anti-CaM monoclonal antibody(McAb) specifically bound to the 17 kD band of expression product. rhCaM was purified by Phenyl-sepharose CL-4B affinity chromatography from recombinant bacterial lysate. 3-4 mg of the purified protein were obtained from 1 liter of bacterial culture. The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM (Sigma). K562 cells and SP2/0 cells were seeded in 24-well or 96-well plate and cultured for 48 h with rhCaM and CaM-antagonist trifluoperazine(TFP). Cell proliferation rates was determined by MTT assay. There was a significant positive correlation between the concentrations of rhCaM and the cell proliferation rates. CaM-antagonist TFP had an inhibitory effect on cell proliferation rate. The inhibition could be corrected by the addition of extracellular rhCaM.     

cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination

The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.     

Ubiquitin ligase substrate identification through quantitative proteomics at both the protein and peptide levels

Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.     

Ubiquitination and proteasome-dependent degradation of human eukaryotic translation initiation factor 4E

Translation initiation factor 4E (eIF4E) is a cytoplasmic cap-binding protein that is required for cap-dependent translation initiation. Here, we have shown that eIF4E is ubiquitinated primarily at Lys-159 and incubation of cells with a proteasome inhibitor leads to increased eIF4E levels, suggesting the proteasome-dependent proteolysis of ubiquitinated eIF4E. Ubiquitinated eIF4E retained its cap binding ability, whereas eIF4E phosphorylation and eIF4G binding were reduced by ubiquitination. The W73A mutant of eIF4E exhibited enhanced ubiquitination/degradation, and 4E-BP overexpression protected eIF4E from ubiquitination/degradation. Because heat shock or the expression of the carboxyl terminus of heat shock cognate protein 70-interacting protein (Chip) dramatically increased eIF4E ubiquitination, Chip may be at least one ubiquitin E3 ligase responsible for eIF4E ubiquitination.     

Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint

The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.