Filamins but Not Janus Kinases Are Substrates of the ASB2α Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells

The ASB2α protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2α substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2α biological effects is crucial for the understanding of ASB2α functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2α induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates.     

E3 Ubiquitin Ligases in Protein Quality Control Mechanism

In living cells, polypeptide chains emerging from ribosomes and preexisting polypeptide chains face constant threat of misfolding and aggregation. To prevent protein aggregation and to fulfill their biological activity, generally, protein must fold into its proper three-dimensional structure throughout their lifetimes. Eukaryotic cell possesses a quality control (QC) system to contend the problem of protein misfolding and aggregation. Cells achieve this functional QC system with the help of molecular chaperones and ubiquitin-proteasome system (UPS). The well-conserved UPS regulates the stability of various proteins and maintains all essential cellular function through intracellular protein degradation. E3 ubiquitin ligase enzyme determines specificity for degradation of certain substrates via UPS. New emerging evidences have provided considerable information that various E3 ubiquitin ligases play a major role in cellular QC mechanism and principally designated as QC E3 ubiquitin ligases. Nevertheless, very little is known about how E3 ubiquitin ligase maintains QC mechanism against abnormal proteins under various stress conditions. Here in this review, we highlight and discuss the functions of various E3 ubiquitin ligases implicated in protein QC mechanism. Improving our knowledge about such processes may provide opportunities to modulate protein QC mechanism in age-of-onset diseases that are caused by protein aggregation.     

Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein

It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to 125I-lysozyme, the conjugates produced are very tightly bound to E3, as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein. In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2, a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2.E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.     

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.     

A ubiquitin-protein ligase specific for type III protein substrates

A previously studied species of ubiquitin-protein ligase contains specific sites for the binding of basic (Type I) and bulky hydrophobic (Type II) NH2-terminal amino acid residues of protein substrates. We now describe another enzyme that ligates ubiquitin specifically to proteins that have NH2-terminal residues other than the above two categories (Type III substrates). The new species of ligase, that we call E3 beta, is separable from the formerly described ligase (termed E3 alpha) by affinity chromatography on protein substrate columns. E3 beta was partially purified from extracts of rabbit reticulocytes and was shown to be required for the breakdown of Type III proteins. Apart from its different substrate specificity, it resembles E3 alpha in some physical properties, in a requirement for ubiquitin carrier protein (E2) for conjugate formation, and in its action to ligate multiple ubiquitin units to the substrate protein. The denatured derivative of bovine pancreatic ribonuclease is a specific substrate for E3 alpha, while that of ribonuclease S-protein is a good substrate for E3 beta. Since S-protein is formed by the removal from ribonuclease of NH2-terminal S-peptide, it is suggested that E3 beta interacts with an NH2-terminal determinant exposed in ribonuclease S-protein.     

In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation

Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.     

The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.     

Alternative Reading Frame Supports An Alternative Model for Retinoblastoma

The ubiqutin-proteasome system is the major pathway by which cells target proteins for degradation in a specific manner. The E3 ubiquitin ligase, which brings targeted proteins (substrates) and activated ubiquitin in close proximity, enabling covalent conjugation of ubiquitin to the substrate, is an essential component of this system. Of the E3 ligases, the cullin (CUL) ligases are of high interest because of their capacity to form multiple distinct E3 complexes to ubiquitinate a potentially large number of substrates. Of the six closely related cullins, very little is known about how specific substrates are recruited to CUL4-dependent ligases. A recent paper in Nature Cell Biology may shed some light on this issue as well as on the function of DDB1, a damaged-DNA binding protein that has long been associated with DNA repair.     

Molecular dynamics simulations to investigate the effects of zinc ions on the structural stability of the c-Cbl RING domain

In eukaryotic cells, ubiquitylation of proteins plays a critical role in regulating diverse cell processes by the ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin protein ligase (E3). E3 is the key component that confers specificity to ubiquitylation and directs the conjugation of ubiquitin to a specific target protein. RING domains are small structured protein domains that require the coordination of zinc ions for a stable tertiary fold and some of them are involved in the E3 family. In this study, we reported the detailed relationships between the two zinc ions and the structural stability of the c-Cbl RING domain by molecular dynamics simulations. Our results show that these two zinc ions play an important role in maintaining both the secondary and tertiary structural stabilities of the c-Cbl RING domain. Our results also reveal that the secondary structural stability of the c-Cbl RING domain is mainly determined by the hydrogen-bonding networks in or near the two zinc ion binding sites. Our results further demonstrate that zinc ion binding site 2 is more structurally stable than site 1.     

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of ～1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed.     

Parkin facilitates the elimination of expanded polyglutamine proteins and leads to preservation of proteasome function

Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated alpha-synuclein, Pael-R, and synphilin-1. We now show that parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Using a cellular proteasome indicator system based on a destabilized form of green fluorescent protein, we demonstrate that parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome, which may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. We speculate that parkin may function to relieve endoplasmic reticulum stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's disease.     

A novel protein modification pathway related to the ubiquitin system.

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.     

泛素连接酶的结构与功能研究进展

泛素化是体内蛋白质翻译后重要修饰之一,是蛋白质降解的信号.泛素连接酶E3是泛素化过程中的关键酶之一,介导活化的泛素从结合酶E2转移到底物,不同的泛素连接酶作用于不同的底物蛋白,决定了泛素化修饰的特异性.根据结构与功能机制的不同,可将泛素连接酶E3分为HECT (homologousto E6AP C terminus)家族和RING-finger家族,前者含有HECT结构域,可直接与泛素连接再将其传递给底物.RING-finger家族的E3发现较晚,庞大且功能复杂,是近年来研究的热点,此家族均包含相似的E2结合结构域和特异的底物结合部分,作为桥梁将活化的泛素从E2直接转移到靶蛋白,其本身并不与泛素发生作用.总结了这2种E3连接酶家族成员的三维结构及功能机制研究的最新进展.     

The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction

The ubiquitin system of intracellular protein degradation controls the abundance of many critical regulatory proteins. Specificity in the ubiquitin system is determined largely at the level of substrate recognition, a step that is mediated by E3 ubiquitin ligases. Analysis of the mechanisms of phosphorylation directed proteolysis in cell cycle regulation has uncovered a new class of E3 ubiquitin ligases called SCF complexes, which are composed of the subunits Skp1, Rbx1, Cdc53 and any one of a large number of different F-box proteins. The substrate specificity of SCF complexes is determined by the interchangeable F-box protein subunit, which recruits a specific set of substrates for ubiquitination to the core complex composed of Skp1, Rbx1, Cdc53 and the E2 enzyme Cdc34. F-box proteins have a bipartite structure--the shared F-box motif links F-box proteins to Skp1 and the core complex, whereas divergent protein-protein interaction motifs selectively bind their cognate substrates. To date all known SCF substrates are recognised in a strictly phosphorylation dependent manner, thus linking intracellular signalling networks to the ubiquitin system. The plethora of different F-box proteins in databases suggests that many pathways will be governed by SCF-dependent proteolysis. Indeed, genetic analysis has uncovered roles for F-box proteins in a variety of signalling pathways, ranging from nutrient sensing in yeast to conserved developmental pathways in plants and animals. Moreover, structural analysis has revealed ancestral relationships between SCF complexes and two other E3 ubiquitin ligases, suggesting that the combinatorial use of substrate specific adaptor proteins has evolved to allow the regulation of many cellular processes. Here, we review the known signalling pathways that are regulated by SCF complexes and highlight current issues in phosphorylation dependent protein degradation.     

SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.     

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.     

The APC11 RING-H2 finger mediates E2-dependent ubiquitination

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.     

A Structural Element within the HUWE1 HECT Domain Modulates Self-ubiquitination and Substrate Ubiquitination Activities

E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and regulate numerous cellular processes. The HECT class of E3 ubiquitin (Ub) ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates. The catalytic domain of HECT Ub ligases has a bilobal architecture that separates the E2 binding region and catalytic site. An important question regarding HECT domain function is the control of ligase activity and specificity. Here we present a functional analysis of the HECT domain of the E3 ligase HUWE1 based on crystal structures and show that a single N-terminal helix significantly stabilizes the HECT domain. We observe that this element modulates HECT domain activity, as measured by self-ubiquitination induced in the absence of this helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle changes to the protein may be at the heart of the vast spectrum of substrate specificities displayed by HECT domain E3 ligases.     

E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer

During ubiquitin ligation, an E2 conjugating enzyme receives ubiquitin from an E1 enzyme and then interacts with an E3 ligase to modify substrates. Competitive binding experiments with three human E2-E3 protein pairs show that the binding of E1s and of E3s to E2s are mutually exclusive. These results imply that polyubiquitination requires recycling of E2 for addition of successive ubiquitins to substrate.     

Inhibition of E6-induced degradation of its cellular substrates by novel blocking peptides

The E6 oncoprotein derived from the tumour-associated human papillomavirus (HPV) types induces the ubiquitin-mediated degradation of several cellular proteins by conjugating them with the cellular ubiquitin ligase E6-AP. This is a HECT domain-containing ligase that was originally identified through its involvement in the E6-mediated degradation of the cellular tumour suppressor protein p53. Here we have investigated, in more detail, the nature of the E6/E6-AP interaction using binding peptides isolated from an E6-specific library. The selected peptides were either predicted or shown to have an alpha-helical core resembling the E6-binding motif on E6-AP, as well as amino acid alterations that increased their affinity for E6. These peptides were potent inhibitors of the E6/E6-AP interaction. Further analysis of the effects of these peptides on the ability of E6 to direct the proteolytic degradation of its various substrates, including p53, Dlg and the MAGI family of proteins, as well as using E6-AP immunodepletion, revealed striking differences in the mechanism by which E6 targets its cellular substrates for degradation. These results suggest that the site on E6 bound by E6-AP is also most likely occupied by other, as yet unidentified, ubiquitin ligases.