Ubiquitin Ligase Function Research Articles

Biophysical characterization of elongin C from Saccharomyces cerevisiae.

Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.

Playing Tag with HIF: The VHL Story.

Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL is the cause of inherited VHL disease and is associated with sporadic kidney cancer. pVHL is found in a multiprotein complex with elongins B/C, Cul2, and Rbx1 forming an E3 ubiquitin ligase complex called VEC. This modular enzyme targets the α subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction. Consequently, tumour cells lacking functional pVHL overproduce the products of HIF-target genes such as vascular endothelial growth factor (VEGF), which promotes angiogenesis. This likely accounts for the hypervascular nature of VHL-associated neoplasms. Although pVHL has been linked to the cell-cycle, differentiation, and the regulation of extracellular matrix assembly, microenvironment pH, and tissue invasiveness, this review will focus on the recent insights into the molecular mechanisms governing the E3 ubiquitin ligase function of VEC.

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.

The ubiquitin-proteasome pathway: on protein death and cell life.

The discovery of the ubiquitin pathway and its many substrates and functions has revolutionized our concept of intracellular protein degradation. From an unregulated, non‐specific terminal scavenger process, it has become clear that proteolysis of cellular proteins is a highly complex, temporally controlled and tightly regulated process which plays important roles in a broad array of basic cellular processes. It is carried out by a complex cascade of enzymes and displays a high degree of specificity towards its numerous substrates. Among these are cell cycle and growth regulators, components of signal transduction pathways, enzymes of house keeping and cell‐specific metabolic pathways, and mutated or post‐translationally damaged proteins. The system is also involved in processing major histocompatibility complex (MHC) class I antigens. For many years it has been thought that activity of the system is limited to the cytosol and probably to the nucleus. However, recent experimental evidence has demonstrated that membrane‐anchored and even secretory pathway‐compartmentalized proteins are also targeted by the system. These proteins must be first translocated in a retrograde manner into the cytosol, as components of the pathway have not been identified in the endoplasmic reticulum (ER) lumen. With the multiple cellular targets, it is not surprising that the system is involved in the regulation of many basic cellular processes such as cell cycle and division, differentiation and development, the response to stress and extracellular modulators, morphogenesis of neuronal networks, modulation of cell surface receptors, ion channels and the secretory pathway, DNA repair, regulation of the immune and inflammatory responses, biogenesis of organelles and apoptosis. One would also predict that aberrations in such a complex system may be implicated in the pathogenesis of many diseases, both inherited and acquired. Recent evidence shows that this is indeed the case. Degradation of a protein by the ubiquitin system involves two distinct …