Post-translational modification of proteins by covalent attachment of a small protein ubiquitin or a polymeric chain of ubiquitin molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, the monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one ubiquitin and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in the chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between the two adjacent ubiquitin monomers. By contrast, K63-linked chains, which act as a non-proteolytic, regulatory signal, adopt an extended conformation that lacks the hydrophobic inter-ubiquitin contact. Little is known about functional roles of the so-called “non-canonical” chains, linked via K6, K11, K27, K29, K33, or head-to-tail; and no structural information on these chains is available, except for the crystal structure of the head-to-tail linked diubiquitin. In this study, we use molecular modeling to examine whether any of the non-canonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible di-ubiquitin chains can be divided into two groups: K6-, K11-, K27-, and K48-linked chains are predicted to form a closed conformation, whereas chains linked via K29, K33, K63, or head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail linked chains. Our study also predicts structural models for di-ubiquitins linked via K6, K11, 3 and K27. Implications of these findings for linkage-selective recognition of the non-canonical polyubiquitin signals by various receptors are discussed.
polyubiquitin; non-canonical linkage; isopeptide linkage; head-to-tail; modeling
Lys48-linked tetraubiquitin, hexaubiquitin and octaubiquitin were enzymatically synthesized, purified and crystallized. X-ray diffraction data sets for tetraubiquitin and hexaubiquitin were collected at 1.6 and 1.8 Å resolution, respectively.
Post-translational modification of proteins by covalent attachment of ubiquitin regulates diverse cellular events. A Lys48-linked polyubiquitin chain is formed via an isopeptide bond between Lys48 and the C-terminal Gly76 of different ubiquitin molecules. The chain is attached to a lysine residue of a substrate protein, which leads to proteolytic degradation of the protein by the 26S proteasome. In order to reveal the chain-length-dependent higher order structures of polyubiquitin chains, Lys48-linked polyubiquitin chains were synthesized enzymatically on a large scale and the chains were separated according to chain length by cation-exchange column chromatography. Subsequently, crystallization screening was performed using the hanging-drop vapour-diffusion method, from which crystals of tetraubiquitin, hexaubiquitin and octaubiquitin chains were obtained. The crystals of the tetraubiquitin and hexaubiquitin chains diffracted to 1.6 and 1.8 Å resolution, respectively. The tetraubiquitin crystals belonged to space group C2221, with unit-cell parameters a = 58.795, b = 76.966, c = 135.145 Å. The hexaubiquitin crystals belonged to space group P21, with unit-cell parameters a = 51.248, b = 102.668, c = 51.161 Å. Structural analysis by molecular replacement is in progress.
ubiquitin; Lys48-linked polyubiquitin
Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as non-proteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, in order to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.
ubiquitin; polyubiquitin; ubiquitin interacting motif; S5a; chemical shift perturbation mapping; ligand binding
Ubiquitin is a highly conserved 76-amino acid polypeptide that is found throughout the eukaryotic kingdom. The covalent conjugation of ubiquitin (often in the form of polymers) to substrates governs a variety of biological processes ranging from proteolysis to DNA damage tolerance. The functional flexibility of this post-translational modification roots in the existence of a large number of ubiquitinating enzymes that catalyze the formation of distinct ubiquitin polymers, which in turn encode different signals. This review summarizes the recent advances in the field with an emphasis on the non-canonical functions of polyubiquitination. We also discuss the potential mechanism of chain linkage specification as well as how structural disparity in ubiquitin polymers may be distinguished by ubiquitin receptors to translate the versatile ubiquitin signals into various cellular functions.
ubiquitin; polyubiquitination; p97; UBA; proteasome; autophagy
Protein ubiquitination is a highly conserved, central mechanism to regulate cellular events in all eukaryotes, such as proteasomal degradation, protein trafficking, DNA repair, synaptic plasticity and immune response. The consequence of protein ubiquitination is modulated by the structure of ubiquitin moiety attached on the substrates, including ubiquitin monomer and diverse polyubiquitin chains with different linkages (N-terminus, K6, K11, K27, K29, K33, K48 and K63). The development of ubiquitin-enrichment strategies coupled with sensitive mass spectrometry enables direct analysis of ubiquitinated proteins in cells, providing an invaluable tool for ubiquitin research. In this chapter we describe recent technology updates for analyzing tissue-specific ubiquitin conjugates in transgenic models, as well as targeted proteomics methods for quantifying different polyubiquitin chain linkages in any type of samples, including human tissues.
ubiquitin; proteomics; mass spectrometry; tissue
The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like (UbL) domain. Thus, arrangement and location of the proteasome initiation region affect a protein’s fate and play a central role in selecting proteins for proteasome-mediated degradation.
The ubiquitin conjugation system regulates a wide variety of biological phenomena, including protein degradation and signal transduction, by regulating protein function via polyubiquitin conjugation in most cases. Several types of polyubiquitin chains exist in cells, and the type of polyubiquitin chain conjugated to a protein seems to determine how that protein is regulated. We identified a novel linear polyubiquitin chain and the ubiquitin-protein ligase complex that assembles it, designated LUBAC. Both were shown to have crucial roles in the canonical NFκB activation pathway. This year, three groups, including our laboratory, identified SHARPIN as a new subunit of LUBAC. Of great interest, Sharpin was identified as a causative gene of chronic proliferative dermatitis in mice (cpdm), which is characterized by numerous inflammatory symptoms including chronic dermatitis, arthritis and immune disorders. Deletion of SHARPIN drastically reduces the amount of LUBAC and attenuates signal-induced NFκB activation. The pleomorphic symptoms of cpdm mice suggest that LUBAC-mediated NFκB activation may play critical roles in mammals and be involved in various disorders. A forward look into the linear polyubiquitin research is also discussed.
ubiquitin; linear ubiquitination; NFκB; LUBAC; SHARPIN; cpdm; chronic dermatitis; TNFα
Ubiquitination involves the covalent attachment of the ubiquitin C-terminus to the lysine sidechain of a substrate protein by an isopeptide bond. The modification can comprise a single ubiquitin moiety or a chain of ubiquitin molecules joined by isopeptide bonds between the C-terminus of one ubiquitin with one of the seven lysine residues in the next ubiquitin. Modification of substrate proteins with Lys63-linked polyubiquitin plays a key non-degradative signaling role in many biological processes including DNA repair and NF-κB activation, whereas substrates modified by lysine-48 (Lys48) linked chains are targeted to the proteasome for degradation. The distinct signaling properties of alternatively linked ubiquitin chains presumably stems from structural differences that can be distinguished by effector proteins. We have determined the crystal structure of Lys63 tetraubiquitin at a resolution of 1.96 Å and performed Small Angle X-ray scattering (SAXS) experiments and molecular dynamics (MD) simulations to probe the conformation of Lys63 tetraubiquitin in solution. The chain adopts a highly extended conformation in the crystal, in contrast with the compact globular fold of Lys48 Ub4. Small Angle X-ray scattering (SAXS) experiments show that the tetraubiquitin chain is dynamic in solution, adopting an ensemble of conformations that are more compact than the extended form in the crystal. The results of these studies provide a basis for understanding the differences in the behavior and recognition of Lys63 polyubiquitin chains.
Polyubiquitin is a diverse signal both in terms of chain length and linkage type. Lysine48-linked ubiquitin is essential for marking targets for proteasomal degradation but the significance and relative abundance of different linkages remain ambiguous. Here we dissect the relationship of two proteasome-associated polyubiquitin-binding proteins, Rpn10 and Dsk2, and demonstrate how Rpn10 filters Dsk2 interactions, maintaining proper function of the ubiquitin-proteasome system. Using quantitative mass spectrometry of ubiquitin, we found that in S. cerevisiae under normal growth conditions the majority of conjugated ubiquitin was linked via lysine48 and lysine63. In contrast, upon DSK2 induction, conjugates accumulated primarily in the form of lysine48-linkages correlating with impaired proteolysis and cytotoxicity. By restricting Dsk2 access to the proteasome, extraproteasomal Rpn10 was essential for alleviating the cellular stress associated with Dsk2. This work highlights the importance of polyubiquitin shuttles such as Rpn10 and Dsk2 in controlling the ubiquitin landscape.
Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This however has been a major impediment in the ubiquitin field. Here we present a robust, efficient, and widely accessible method for controlled iterative non-enzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the non-enzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked di-ubiquitin, and a mixed-linkage Lys33,Lys11-linked tri-ubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.
Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.
As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone, and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin-chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly-tagged cargo. We propose a mechanism for a malleable ubiquitin-signal that depends both on chain-length and combination of receptors to produce tetra-ubiquitin as an efficient signal threshold.
Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13-Å pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.
The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling.
analytical ultracentrifugation (AUC); IKK (inhibitory κB kinase) complex; isothermal titration calorimetry (ITC); multi-angle light scattering (MALS); protein–protein interaction; ubiquitin; AUC, analytical ultracentrifugation; CoZi, C-terminal portion of NEMO encompassing the second coiled-coil region and the leucine zipper domain; IκB, inhibitory κB; IKK, IκB kinase; IKKNBD, IKKβ NEMO-binding domain; ITC, isothermal titration calorimetry; LZ, leucine zipper; MALS, multi-angle laser light scattering; NF-κB, nuclear factor κB; NEMO, NF-κB essential modulator; NEMO355, NEMO residues 1–355, with mutations C54S and K285N; SEC, size-exclusion chromatography; TCEP, tris-(2-carboxyethyl)phosphine; RI, refractive index; RIP1, receptor interaction protein kinase; vFLIP, viral FLICE [FADD (Fas-associated death domain)-like interleukin 1β-converting enzyme]-inhibitory protein; ZF, zinc finger
Newly synthesized membrane proteins are queried by ubiquitin ligase complexes and triaged between degradative and nondegradative fates. The mechanisms that convert modest differences in substrate-ligase interactions into decisive outcomes of ubiquitination are not well understood. Here, we reconstitute membrane protein recognition and ubiquitination in liposomes using purified components from a viral-mediated degradation pathway. We find that substrate-ligase interactions in the membrane directly influence processivity of ubiquitin attachment to modulate polyubiquitination. Unexpectedly, differential processivity alone could not explain the differential fates in cultured cells of degraded and nondegraded clients. Both computational and experimental analyses identified continuous deubiquitination as a prerequisite for maximal substrate discrimination. Deubiquitinases reduce polyubiquitin dwell times preferentially on clients that dissociate more rapidly from the ligase. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in net degradation. These results provide a conceptual framework for substrate discrimination during membrane protein quality control.
•Membrane protein ubiquitination has been reconstituted with purified factors in vitro•Differential ligase interactions alone cannot explain how clients are discriminated•Maximal client discrimination requires competing deubiquitination activity•Deubiquitinases control the dwell time of a degradation mark on potential clients
Optimal substrate discrimination by the protein degradation machinery requires deubiquitinases (DUBs) to counteract E3 ligases to exaggerate the differential polyubiquitination among potential clients. DUBs have greater access to well-folded substrates that show weaker interactions with the ligase, protecting them from degradation.
A new crystal form of Lys48-linked diubiquitin was obtained and its structure was determined by X-ray crystallography to 1.6 Å resolution.
Lys48-linked polyubiquitin chains are recognized by the proteasome as a tag for the degradation of the attached substrates. Here, a new crystal form of Lys48-linked diubiquitin (Ub2) was obtained and the crystal structure was refined to 1.6 Å resolution. The structure reveals an ordered isopeptide bond in a trans configuration. All three molecules in the asymmetric unit were in the same closed conformation, in which the hydrophobic patches of both the distal and the proximal moieties interact with each other. Despite the different crystallization conditions and different crystal packing, the new crystal structure of Ub2 is similar to the previously published structure of diubiquitin, but differences are observed in the conformation of the flexible isopeptide linkage.
ubiquitin; diubiquitin; Lys48; isopeptide bonds; UBA
RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response.
Polyubiquitin chains are regulatory signals for a wide array of biological processes. Recent structural studies reveal novel modes of polyubiquitin chain recognition and implicate the diverse repertoire of interactions in providing the specificity of polyubiquitin recognition.
Polyubiquitin chains are regulatory signals for a wide array of biological processes. Recent structural studies reveal novel modes of polyubiquitin chain recognition and implicate the diverse repertoire of interactions in providing the specificity of polyubiquitin recognition.
Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular processes. TAK1, a member of the MAPKKK family, is essential for TNFα-induced NF-κB activation. Phosphorylation and Lys63-linked polyubiquitination (polyUb) of TAK1 are critical for its activation. However, whether TAK1 is regulated by polyubiquitination-mediated protein degradation after its activation remains unknown. Here we report that TNFα induces TAK1 Lys48 linked polyubiquitination and degradation at the later time course. Furthermore, we provide direct evidence that TAK1 is modified by Lys48-linked polyubiquitination at lysine-72 by mass spectrometry. A K72R point mutation on TAK1 abolishes TAK1 Lys48-linked polyubiquitination and enhances TAK1/TAB1 co-overexpression-induced NF-κB activation. As expected, TAK1 K72R mutation inhibits TNFα-induced Lys48-linked TAK1 polyubiquitination and degradation. TAK1 K72R mutant prolongs TNFα-induced NF-κB activation and enhances TNFα-induced IL-6 gene expression. Our findings demonstrate that TNFα induces Lys48-linked polyubiquitination of TAK1 at lysine-72 and this polyubiquitination-mediated TAK1 degradation plays a critical role in the downregulation of TNFα-induced NF-κB activation.
TNFα; TAK1; Lys48-linked polyubiquitination; degradation
NF-κB activation in the TNF, IL-1 and Toll-like receptor pathways requires Lys63-linked non-degradative polyubiquitination. A20 is a specific feedback inhibitor of NF-κB activation in these pathways and possesses dual ubiquitin editing functions. While the N-terminal domain of A20 is a de-ubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-κB activation in the TNF, IL-1 and Toll-like receptor pathways.
A20; crystal structure; de-ubiquitination; DUB; TRAF6
All seven lysine residues in ubiquitin contribute to the synthesis of polyubiquitin chains on protein substrates. Whereas K48-linked chains are well established as mediators of proteasomal degradation, and K63-linked chains act in nonproteolytic events, the roles of unconventional polyubiquitin chains linked through K6, K11, K27, K29, or K33 are not well understood. Here we report that the unconventional linkages are abundant in vivo, and all non-K63 linkages may target proteins for degradation. Ubiquitin with K48 as the single lysine cannot support yeast viability, and different linkages have partially redundant functions. By profiling both the entire yeast proteome and ubiquitinated proteins in wild-type and ubiquitin K11R mutant strains using mass spectrometry, we identified K11 linkage-specific substrates, including Ubc6, a ubiquitin conjugating enzyme involved in endoplasmic reticulum-associated degradation (ERAD). Ubc6 primarily synthesizes K11-linked chains, and K11 linkages function in the ERAD pathway. Thus, unconventional polyubiquitin chains are critical for ubiquitin-proteasome system function.
ubiquitin; polyubiquitin chains; K11 linkages; protein degradation; proteasome; Ubc6; mass spectrometry; proteomics; SILAC
Plants utilize the ubiquitin-proteasome system (UPS) to modulate nearly every aspect of growth and development. Ubiquitin is covalently attached to target proteins through the action of three enzymes known as E1, E2, and E3. The ultimate outcome of this post-translational modification depends on the nature of the ubiquitin linkage and the extent of polyubiquitination. In most cases, ubiquitination results in degradation of the target protein in the 26S proteasome. During the last 10 years it has become clear that the UPS plays a prominent regulatory role in hormone biology. E3 ubiquitin ligases in particular actively participate in hormone perception, de-repression of hormone signaling pathways, degradation of hormone specific transcription factors, and regulation of hormone biosynthesis. It is certain that additional functions will be discovered as more of the nearly 1200 potential E3s in plants are elucidated.
hormone signaling; ligases; ubiquitin-proteasome system
Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site.
Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20 residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of the interactions between UIMs with ubiquitin have shown that UIMs adopt an extended structure of a single α-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, changes in the structure and dynamics of ubiquitin have been characterized upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface as well as global changes in ubiquitin due to UIM binding at the picosecond to nanosecond and microsecond to millisecond protein motions by NMR relaxation. Changes in generalized order parameters of amide groups show a distinct trend toward increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of 15N CPMG relaxation dispersion measurements suggest the presence of two types of motions, one directly related to the UIM-binding interface, the other being induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects in protein motions at timescales ranging from picoseconds to milliseconds.
Ubiquitin Interacting Motif; Ubiquitin Fusion Protein; solution NMR structure; conformational exchange; protein recognition dynamics