泛素融合降解蛋白UFD1的结构与功能

泛素-蛋白酶体途径是细胞内蛋白质选择性降解的主要途径,参与多种真核生物细胞生理过程,与细胞的生理功能和病理状态有着密切的关系。该途径中UFD1作为泛素识别因子介导泛素化的靶蛋白至26S蛋白酶体降解。该文在概述泛素-蛋白酶体途径作用机制的基础上,对哺乳动物和酵母UFD1蛋白的结构及其在细胞周期调控、转录调控、内质网相关蛋白降解中的功能进行了综述。     

The Ubiquitin E3 Ligase NOSIP Modulates Protein Phosphatase 2A Activity in Craniofacial Development

Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans.     

NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI from Degradation by the Proteasome

The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.In eukaryotes, eukaryotic translation initiation factor 4G (eIF4G) plays a central role in the recruitment of ribosomes to the mRNA 5′ end and is therefore critical for the regulation of protein synthesis (14). Two homologues of eIF4G, eIF4GI and eIF4GII, have been cloned (15). Although they differ in various respects, both homologues clearly function in translation initiation. The most thoroughly studied of these is eIF4GI, which serves as a scaffolding protein for the assembly of eIF4F, a protein complex composed of eIF4E (the mRNA cap-binding factor) and eIF4A (an ATP-dependent RNA helicase). Thus, via its association with the mRNA cap-binding protein eIF4E and with another translation initiation factor (eIF3) which is bound to the 40S ribosomal subunit, eIF4GI creates a physical link between the mRNA cap structure and the ribosome, thus facilitating cap-dependent translation initiation (25). eIF4GI functions also in cap-independent, internal ribosome entry site (IRES)-mediated translation initiation. For instance, upon picornavirus infection, eIF4G is rapidly attacked by viral proteases. The resulting eIF4GI cleavage products serve to reprogram the cell''s translational machinery, as the N-terminal cleavage product inhibits cap-dependent translation of host cell mRNAs by sequestering eIF4E while the C-terminal cleavage product stimulates IRES-mediated translation of viral mRNAs (23). Similarly, apoptotic caspases cleave eIF4G into an N-terminal fragment that blocks cap-dependent translation and a C-terminal fragment that is utilized for IRES-mediated translation of mRNAs encoding proapoptotic proteins (22).The regulation of eIF4GI cleavage by viral proteases or apoptotic caspases has been extensively studied. Little is known, however, about the regulation of eIF4GI steady-state levels. Yet the eIF4GI amount that exists at a given moment results from the sum of the effects of de novo synthesis and ongoing degradation. Many cellular proteins are physiologically degraded by the proteasome. This has been shown to be true for eIF4GI, as the factor can be degraded by the proteasome in vitro (5) and in living cells (6). However, how eIF4GI targeting for or protection from destruction by the proteasome is regulated remains unknown.There are two major routes to degradation by the proteasome. In the more conventional route, polyubiquitinated proteins are targeted to the 26S proteasome. Alternatively, a few proteins can be degraded by the 20S proteasome (and sometimes by the 26S proteasome) in a ubiquitin-independent manner (16). Interestingly, it has been shown recently that a few of these proteins (1, 2, 13) can be protected from degradation by the 20S proteasome by binding to the NAD(P)H quinone-oxydoreductase 1 (NQO1). It has been proposed that NQO1 may interact with the 20S proteasome and may consequently block access of target proteins to the 20S degradation core. Because eIF4GI can be degraded in vitro by the 20S proteasome (5) and since it appears that proteasomes can degrade eIF4GI in living cells independently of ubiquitination (6), we asked whether NQO1 could protect eIF4GI from degradation by the proteasome.     

FKBP38 Protects Bcl-2 from Caspase-dependent Degradation

The cellular processes that regulate Bcl-2 at the posttranslational levels are as important as those that regulate bcl-2 synthesis. Previously we demonstrated that the suppression of FK506-binding protein 38 (FKBP38) contributes to the instability of Bcl-2 or leaves Bcl-2 unprotected from degradation in an unknown mechanism. Here, we studied the underlying molecular mechanism mediating this process. We first showed that Bcl-2 binding-defective mutants of FKBP38 fail to accumulate Bcl-2 protein. We demonstrated that the FKBP38-mediated Bcl-2 stability is specific as the levels of other anti-apoptotic proteins such as Bcl-X^L and Mcl-1 remained unaffected. FKBP38 enhanced the Bcl-2 stability under the blockade of de novo protein synthesis, indicating it is posttranslational. We showed that the overexpression of FKBP38 attenuates reduction rate of Bcl-2, thus resulting in an increment of the intracellular Bcl-2 level, contributing to the resistance of apoptotic cell death induced by the treatment of kinetin riboside, an anticancer drug. Caspase inhibitors markedly induced the accumulation of Bcl-2. In caspase-3-activated cells, the knockdown of endogenous FKBP38 by small interfering RNA resulted in Bcl-2 down-regulation as well, which was significantly recovered by the treatment with caspase inhibitors or overexpression of FKBP38. Finally we presented that the Bcl-2 cleavage by caspase-3 is blocked when Bcl-2 binds to FKBP38 through the flexible loop. Taken together, these results suggest that FKBP38 is a key player in regulating the function of Bcl-2 by antagonizing caspase-dependent degradation through the direct interaction with the flexible loop domain of Bcl-2, which contains the caspase cleavage site.     

Phosphorylation Regulates the Ubiquitin-independent Degradation of Yeast Pah1 Phosphatidate Phosphatase by the 20S Proteasome

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state.     

A Label-free Quantitative Proteomics Strategy to Identify E3 Ubiquitin Ligase Substrates Targeted to Proteasome Degradation

The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.The ubiquitin-proteasome system (UPS)¹ plays an essential role in the regulation of protein stability in eukaryotic cells. Degradation of a protein by the UPS entails two successive steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and its degradation by the 26 S proteasome (1, 2). Ubiquitylation of protein substrates occurs through the sequential action of distinct enzymes: a ubiquitin-activating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3, responsible for the specific recognition of substrates. Increasing attention has been recently given to the UPS leading to the identification of hundreds of E3 ubiquitin ligases (E3s). Two major classes of E3s have been described: (i) E3s of the HECT (homologous to the E6-associated protein carboxyl terminus) domain family that function as ubiquitin carriers (3, 4) and (ii) E3s of the RING (really interesting new gene) or of the U box families that have no inherent catalytic activity but recruit an E2 enzyme toward substrates (5–7).Classical approaches to identify substrates of E3s are based on the identification of interacting proteins. Although these have successfully led to the identification of a number of substrates of monomeric E3s, identification of substrates of multimeric E3s is very challenging because of the weak affinity of substrates for their requisite specificity subunit and because of the labile nature of the substrate complexed with the specificity subunit (8).Acute promyelocytic leukemia (APL) is associated with six reciprocal translocations always involving the retinoic acid receptor α (RARα) gene (9–11). The RARα protein is a member of the nuclear receptor superfamily that stimulates myeloid differentiation in the presence of its ligand, all-trans-retinoic acid (RA). In more than 95% of APL, the t(15;17) translocation between the promyelocytic leukemia (PML) gene on chromosome 15 and the RARα gene on chromosome 17 produces the PML-RARα fusion protein (12). The PML-RARα protein enhances the repression of RARα target genes by increasing associations with corepressors (13–15) and by recruiting DNA methyltransferases (16). These complexes dissociate from the PML-RARα fusion protein in the presence of pharmacological concentrations of RA perhaps explaining why APL cells are sensitive to RA treatment. Indeed at pharmacological concentrations, RA induces complete remission in a high percentage of APL patients (17–19). By studying RA-induced differentiation of APL cells we have attempted to identify some of the genes that may be up-regulated during this process to further understand the control of growth and differentiation in leukemia (20). One gene identified in this manner, ASB2 (ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) is an RA-response gene involved in induced differentiation of myeloid leukemia cells (21–23).The ASB2 protein is a subunit of a multimeric E3 ubiquitin ligase of the cullin-RING ligase family (24, 25). The ASB2 suppressor of cytokine signaling box can be divided into a BC box that defines a binding site for the Elongin BC complex and a Cul5 box that determines the binding specificity for Cullin5 (24, 26). Indeed the ASB2 protein, by interacting with the Elongin BC complex, can assemble with a Cullin5/Rbx1 or -2 module to reconstitute an active E3 ubiquitin ligase complex (23–25). Within this complex, the ASB2 protein is the specificity subunit involved in the recruitment of specific substrate(s). Furthermore endogenous ASB2 protein was copurified with ubiquitin ligase activity in RA-treated APL cells suggesting that, during induced differentiation of leukemia cells, the ASB2 protein may target proteins involved in blocking differentiation to destruction by the proteasome machinery (24). We recently identified actin-binding proteins filamin A (FLNa) and filamin B (FLNb) as ASB2 targets and showed that ASB2 triggers ubiquitylation and drives proteasome-mediated degradation of these proteins during RA-induced differentiation of myeloid leukemia cells (23).With the aim to develop a strategy to identify E3 substrates that are degraded by the proteasome, we used an MS approach to identify ASB2 substrates in physiologically relevant settings. Indeed we used label-free quantitative proteomics to identify proteins that are absent or less abundant in cells that express wild-type ASB2 but that accumulate in cells expressing an ASB2 E3 ligase-defective mutant. Application of label-free MS methods that have the advantage to be simple, fast, and cheap enabled the identification of FLNa and FLNb as ASB2 substrates. This study provides a new strategy for the identification of E3 substrates that have to be degraded.     

Deubiquitinase FAM/USP9X Interacts with the E3 Ubiquitin Ligase SMURF1 Protein and Protects It from Ligase Activity-dependent Self-degradation

Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration, and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ubiquitin ligase activity. This modular architecture allows for interactions with other proteins, which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is autoubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taken together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability.     

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI₂ levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI₂, in T1DM mice.     

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. We showed that 10 phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RNA recognition motifs protects against dephosphorylation, suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.     

Hepatocyte DNA Replication Is Abolished by Inhibitors Selecting Protein Phosphatase 2A Rather Than Phosphatase 1

Primary rat hepatocytes exposed to the phosphoprotein phosphatase (PP) inhibitors microcystin-LR and okadaic acid showed extensive surface protrusions and release of cell fragments, like cells in apoptosis. Microinjected microcystin fully reproduced these effects; the calculated intracellular concentration required for 50% effect being about 1 μM. The effects were counteracted by antagonists of calmodulin or of the multifunctional calmodulin-activated protein kinase II. The DNA replication of the epidermal growth factor-stimulated hepatocytes was nearly completely inhibited by okadaic acid at concentrations below those giving overt morphological effects. However, microcystin did not inhibit the DNA replication. Calmodulin antagonists counteracted the effect of okadaic acid on DNA replication. Microinjection of inhibitor-1 and inhibitor-2 (both directed against PP1) had no effect on DNA replication. Based on the known selectivity of okadaic acid for PP type 2A versus that of type 1, and the lack of such selectivity for microcystin, it is concluded that DNA replication is abolished by moderate inhibition of PP2A. Inhibition of PP1 did not impede DNA replication, suggesting that the two major liver phosphatases may have opposite roles in the regulation of hepatocyte DNA replication.     

H2O2 Inhibits ABA-Signaling Protein Phosphatase HAB1

Due to its ability to be rapidly generated and propagated over long distances, H₂O₂ is an important second messenger for biotic and abiotic stress signaling in plants. In response to low water potential and high salt concentrations sensed in the roots of plants, the stress hormone abscisic acid (ABA) activates NADPH oxidase to generate H₂O₂, which is propagated in guard cells in leaves to induce stomatal closure and prevent water loss from transpiration. Using a reconstituted system, we demonstrate that H₂O₂ reversibly prevents the protein phosphatase HAB1, a key component of the core ABA-signaling pathway, from inhibiting its main target in guard cells, SnRK2.6/OST1 kinase. We have identified HAB1 C186 and C274 as H₂O₂-sensitive thiols and demonstrate that their oxidation inhibits both HAB1 catalytic activity and its ability to physically associate with SnRK2.6 by formation of intermolecular dimers.