The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

Perturbing the Ubiquitin Pathway Reveals How Mitosis Is Hijacked to Denucleate and Regulate Cell Proliferation and Differentiation In Vivo

The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed

Molecular Interactions of Ubiquitin and Polyubiquitin with Ubiquitin Binding Domains

Ubiquitin is a small protein that is covalently attached to proteins, either as a single ubiquitin moiety or as polyubiquitin chains. A cascade of enzymatic reactions is required for the isopeptide linkage between the C-terminus of ubiquitin and a lysine residue on a substrate protein or another ubiquitin. Attachment of ubiquitin or polyubiquitin, termed ubiquitination, mediates numerous cellular processes by acting as a versatile signal. The signal transmitted by the tag depends on the nature of the modification, which defines the specificity of the tag for different cellular machinery. This versatility is conferred by the variations in polyubiquitin tags, both in terms of length and lysine-linkage. Polyubiquitin chains can adopt a variety of different conformations based on these variations. The conformational and dynamic properties of the tag may optimize its binding to specific ubiquitin binding domains, therefore committing the target protein to distinct cellular outcomes. A combination of NMR methods are used to study the interaction of several ubiquitin binding domains with Lys48- and Lys63-linked di-ubiquitin, the simplest model of a polyubiquitin chain, to gain insights into polyubiquitin recognition. The di-ubiquitin binding interface with ubiquitin-interacting motifs (UIMs) and ubiquitin-associated domains (UBAs) are mapped. Structural models of the complexes are also presented. The results provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interface. The results also show that the UBA domain of Ede1 preferentially binds to Lys63-linked di-ubiquitin. Structural models of the UBA in complex with Lys48- and Lys63-linked di-ubiquitin are shown. Although ubiquitin is highly conserved in eukaryotes, it is promiscuous with regard to its binding partners, ranging from small molecules to UIM and UBA domains. This study examines the effects of point core leucine to serine mutations on UIM and UBA binding specificity. The results show that these mutations bestow ubiquitin with the ability to discriminate between ubiquitin-receptor proteins. Here, we solved the three-dimensional structure of the L69S Ub mutant in solution by NMR. These mutations have a profound effect on binding specificity while causing subtle changes in the protein's three-dimensional fold and reducing its stability. Modification of a specific lysine located on Ub's hydrophobic surface has been reported to inhibit proteasomal degradation and endocytosis. Here, the effects of mutation to tryptophan at this position are investigated within the context of binding to a proteasomal receptor protein, hHR23A, and an endocytic receptor protein, Ede1

Mapping the interactions between Lys48 and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a.

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 nonproteolytic 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, 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 the 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

Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling.

Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules. The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains. Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes. Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking. Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain. The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units. This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W. J., Jeffrey, L. C., Carson, M., Zhijian, C., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647). Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin. In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined. This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations

Long-lived States to monitor protein unfolding by proton NMR.

International audienceThe relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T(1) and T(2) relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S

Personal Role Management: Overview and a Design Study of Email for University Students

Evidence is accumulating about the difficulties that users have in managing their work using contemporary graphical user interfaces. Current designs offer a hierarchy of folders containing documents and taskbar operations to launch/exit applications. We propose a Personal Role Management strategy that emphasizes management of the multiple roles users have in their professional and personal lives. Each role involves coordination with groups of people and accomplishment of tasks within a schedule. We define Personal Role Management and summarize our earlier work that led to this strategy. This current project focused on understanding how Personal Role Management might improve email for college students. College students often assume distinct and predictable roles. Their student role is structured by the rhythm and interactions of classes, projects and exams. In both their family role and their work role for local companies, they deal with separate groups of people. We describe scenarios of use of a role-based email system, an interface mockup and user reactions. This research suggests that using those roles as a driving component for designing an email interface might address problems identified in our surveys and interviews of college students