A ubiquitin ligase complex assembles linear polyubiquitin chains

The ubiquitin system plays important roles in the regulation of numerous cellular processes by conjugating ubiquitin to target proteins. In most cases, conjugation of polyubiquitin to target proteins regulates their function. In the polyubiquitin chains reported to date, ubiquitin monomers are linked via isopeptide bonds between an internal Lys and a C-terminal Gly. Here, we report that a protein complex consisting of two RING finger proteins, HOIL-1L and HOIP, exhibits ubiquitin polymerization activity by recognizing ubiquitin moieties of proteins. The polyubiquitin chain generated by the complex is not formed by Lys linkages, but by linkages between the C- and N-termini of ubiquitin, indicating that the ligase complex possesses a unique feature to assemble a novel head-to-tail linear polyubiquitin chain. Moreover, the complex regulates the stability of Ub-GFP (a GFP fusion protein with an N-terminal ubiquitin). The linear polyubiquitin chain generated post-translationally may function as a new modulator of proteins.     

Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies

Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical–biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a ¹⁵N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.     

Assembly and characterization of five-arm and six-arm DNA branched junctions.

DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.     

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP-the catalytic subunit of LUBAC-is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.     

Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains

UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.     

The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.     

Galactosylation at side chains of branched cyclodextrins by various beta-galactosidases.

The galactosyl transfer reaction to branched cyclodextrins (CDs) was investigated using lactose as a donor substrate and branched CDs as acceptors by various beta-galactosidases. Bacillus circulans beta-galactosidase synthesized galactosyl transfer products to branched CDs, of which the galactose residues were linked at side chains of branched CDs, not directly at CD rings. Aspergillus oryzae and Penicillium multicolor beta-galactosidases also produced derivatives galactosylated at side chains of branched CDs. The structures of main transgalactosylation products of branched CDs by these beta-galactosidases seem to be different from those by B. circulans beta-galactosidase, judging from the retention times on high performance liquid chromatography.     

K11-linked ubiquitin chains as novel regulators of cell division

Modification of proteins with ubiquitin chains is an essential regulatory event in cell cycle control. Differences in the connectivity of ubiquitin chains are believed to result in distinct functional consequences for the modified proteins. Among eight possible homogenous chain types, canonical Lys48-linked ubiquitin chains have long been recognized to drive the proteasomal degradation of cell cycle regulators, and Lys48 is the only essential lysine residue of ubiquitin in yeast. It thus came as a surprise that in higher eukaryotes atypical K11-linked ubiquitin chains regulate the substrates of the anaphase-promoting complex and control progression through mitosis. We discuss recent findings that shed light on the assembly and function of K11-linked chains during cell division.     

Non-enzymatic synthesis of ubiquitin chains: Where chemistry makes a difference

Ubiquitination is a highly important posttranslational modification in eukaryotic cells where a target protein is conjugated to ubiquitin or a chain of ubiquitins via an isopeptide bond to trigger various cellular events such as proteasomal degradation. Rigorous investigations of the ubiquitin signal at the molecular level require homogeneous samples of ubiquitin chains in their free form or as anchored to a protein substrate in adequate quantities. The complexity of ubiquitin chains in terms of linkage types (owing to presence of seven Lys in ubiquitin) makes them difficult to prepare via enzymatic methods. Even more challenging is the attachment of these chains to a protein target at a selected site. This dearth is being filled by the recent developments of novel chemical tools that offer atomic level control over the synthesis for structural and functional studies. These emerging chemical approaches are discussed in this mini-review with focus on the preparation of ubiquitin chains to aid the ongoing efforts in understanding their role in the ubiquitin signal.     

Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function.

In the present work, a method based on an epitope-tagged ubiquitin derivative is described that allows for the unambiguous detection of ubiquitin-protein conjugates formed in vivo or in vitro. Expression in the yeast Saccharomyces cerevisiae of ubiquitin that has been tagged at its amino terminus with a peptide epitope results in the formation of tagged ubiquitin-protein conjugates that are detectable by immunoblotting with a monoclonal antibody that recognizes the tag. The expression of tagged ubiquitin has no adverse effect on vegetative growth and, moreover, can suppress the stress-hypersensitive phenotype of yeast lacking the polyubiquitin gene UBI4. We also show that tagged ubiquitin is correctly conjugated in vivo and in vitro to a short-lived test protein and can be covalently extended into the multimeric ubiquitin chain that is normally required for the degradation of this protein. Surprisingly, however, conjugation of tagged ubiquitin inhibits proteolysis. These and related results suggest that the amino-terminal region of ubiquitin is important in protease-substrate recognition and that the multiubiquitin chain is a dynamic transient structure. The potential of tagged ubiquitin for the identification and isolation of ubiquitin-protein conjugates and ubiquitin-related enzymes, and as a tool in mechanistic studies is discussed.     

Curvature properties of novel forms of phosphatidylcholine with branched acyl chains.

We studied the properties of a series of phosphatidylcholine molecules with branched acyl chains. These lipids have previously been shown to have marked stimulatory effects on the side-chain cleavage activity of cytochrome P450SCC (CYP11A1), an enzyme of the inner mitochondrial membrane. The synthetic lipids used were diacyl phosphatidylcholines with the decanoyl, dodecanoyl or tetradecanoyl chain having a hexyl, octyl or decyl straight chain aliphatic branch at the 2-position. All three lipids lowered the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine, the lipids with longer acyl chains being more effective in this regard. As pure lipids all of the forms were found by X-ray diffraction to be predominantly in the hexagonal phase (HII) over the entire temperature range of 7-75 degrees C. The properties of the HII phase were unusual with regard to the small size of the lattice spacings and the small temperature dependence of the spacings. We used tetradecane to relieve hydrocarbon packing constraints to determine the intrinsic radius of curvature of the lipid monolayer. The elastic bending modulus was measured in the presence of tetradecane by introducing an osmotic gradient across the hexagonal phase cylinders with aqueous solutions of poly(ethylene glycol). The elastic bending modulus was found to be higher than that observed with other lipids and to increase with temperature. Both the small intrinsic radius of curvature and the high elastic bending modulus indicate that the presence of these lipids in bilayer membranes will impose a high degree of negative curvature strain.     

Transketolase inhibitors based on disulfide derivatives of oxythiamine with branched hydrocarbon chains

The experiments on mice have shown that oxythiamine disulphide derivatives with the branched hydrocarbon chains are less toxic in the organism as compared to oxythiamine and corresponding disulphides with the unbranched hydrocarbon chains and also induce a more pronounced inhibition of transketolase in the liver and other tissues. It is found that under the effect of the above substances the recovery of enzymic activity is slower than in the case with the oxythiamine application.     

Increase of asparagine-linked oligosaccharides with branched outer chains caused by cell transformation

By hydrazinolysis, oligosaccharides were released from fucose-labeled glycopeptides obtained from normal and polyoma-transformed baby hamster kidney cells, and their structures were comparatively analyzed. The oligosaccharides have the following structures, with different number of sialyl-galactosyl-N-acetylglucosaminyl outer chains: (±Siaα→Galβ→GlcNAcβ→)_n(Manα→)₂Manβ→GlcNAcβ→(Fucα→)GlcNAc, (in normal cells, n=2, 3 and 4, while in polyoma-transformed cells, n=2,3,4,5 and 6). Transformed cells are relatively rich in oligosaccharides with highly branched outer chains, as compared to normal cells.     

Assembly and secretion of fibrinogen. Degradation of individual chains.

Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.     

Generation of free ubiquitin chains is up-regulated in stress and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5

Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalyzed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.     

Reversible thermal transition of soluble branched chains from slightly acid-treated potato starch

The reversible thermal transition of soluble branched starch chains prepared from slightly acid-treated potato starch granules (ATS) was investigated. Potato starch was immersed in 15% sulfuric acid to obtain ATS with a 1% hydrolysis rate. About half of the molecules of ATS, which spontaneously formed large aggregates with a mass of a few million daltons in aqueous solution, was fractionated and soluble branched starch chains with a relative molecular weight (Mr) of 8.91 x 10(4) were obtained. Structural analysis indicated that the soluble branched starch chains consisted of three unit chains with Mr 7,900 and 21 unit chains with Mr 2,700. DSC and FT-IR measurements showed that the soluble branched starch chains underwent a reversible thermal transition, which is considered to be a helix-coil transition, during heating and cooling, but a debranched sample and beta-limit dextrins showed substantially different thermal behavior, indicating the contribution of the ordered structure of the branched chains.     

Synthesis, calorimetry, and X-ray diffraction of lecithins containing branched fatty acid chains

Lecithins with branched fatty acid chains were synthesized and characterized by differential scanning calorimetry and X-ray diffraction. The influence of three chemical alterations on the phase transition parameters were investigated: length of the branches in 2-position of the acyl chains, position of the branches in the acyl chains, and position of the branched fatty acid chains in the glycerol backbone. The results show that the branched phosphatidylcholines (PCs) have a reduced gel-to-liquid-crystalline phase transition temperature (Tm) compared to the corresponding straight-chain PCs. Depending on both the length of the branches in 2-position of the acyl chains and the position of the branches in the acyl chains, the Tm-values pass through a minimum. The systematic change of the main-transition temperatures Tm is connected with a modified structural polymorphism. If the length of the branches increases three types of polymorphism were observed.     

Alpha-synuclein and parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.