Tommer Ravid

Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways

The yeast Doa10 ubiquitin (Ub) ligase resides in the endoplasmic reticulum (ER)/nuclear envelope (NE), where it functions in ER‐associated degradation (ERAD). Doa10 substrates include non‐ER proteins such as the transcription factor Matα2. Here, we expand the range of Doa10 substrates to include a defective kinetochore component, a mutant NE membrane protein, and a substrate‐regulated human ER enzyme. For all these substrates, Doa10 requires two Ub‐conjugating enzymes, Ubc6 and Ubc7, as well as the Ubc7 cofactor Cue1. Based on a novel genomic screen of a comprehensive gene deletion library and other data, these four proteins appear to be the only nonessential and nonredundant factors generally required for Doa10‐mediated ubiquitination. Notably, the Cdc48 ATPase facilitates degradation of membrane‐embedded Doa10 substrates, but is not required for any tested soluble Doa10 substrates. This distinction is maintained even when comparing membrane and soluble proteins bearing the same degradation signal. Thus, while Doa10 ubiquitinates both membrane and soluble proteins, the mechanisms of subsequent proteasome targeting differ.

Epidermal growth factor receptor exposed to oxidative stress undergoes Src- and caveolin-1-dependent perinuclear trafficking

The epidermal growth factor (EGF) receptor (EGFR) has been found to be overexpressed in several types of cancer cells, and the regulation of its oncogenic potential has been widely studied. The paradigm for EGFR down-regulation involves the trafficking of activated receptor molecules from the plasma membrane, through clathrin-coated pits, and into the cell for lysosomal degradation. We have previously shown that oxidative stress generated by H2O2 results in aberrant phosphorylation of the EGFR. This leads to the loss of c-Cbl-mediated ubiquitination of the EGFR and, consequently, prevents its degradation. However, we have found that c-Cbl-mediated ubiquitination is required solely for degradation but not for internalization of the EGFR under oxidative stress. To further examine the fate of the EGFR under oxidative stress, we used confocal analysis to show that the receptor not only remains co-localized with caveolin-1 at the plasma membrane, but at longer time points, is also sorted to a perinuclear compartment via a clathrin-independent, caveolae-mediated pathway. Our findings indicate that although the EGFR associates with caveolin-1 constitutively, caveolin-1 is hyperphosphorylated only under oxidative stress, which is essential in transporting the EGFR to a perinuclear location, where it is not degraded and remains active. Thus, oxidative stress may have a role in tumorigenesis by not only activating the EGFR but also by promoting prolonged activation of the receptor both at the plasma membrane and within the cell.

Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue

Cells have quality-control mechanisms to recognize non-native protein structures and either help the proteins fold or promote their degradation. Ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) work together to assemble polyubiquitin chains on misfolded or misassembled proteins, which are then degraded by the proteasome. Here, we find that Ubc7, a yeast E2, can itself undergo degradation when its levels exceed that of its binding partner Cue1, a transmembrane protein that tethers Ubc7 to the endoplasmic reticulum. Unassembled, and thus mislocalized, Ubc7 is targeted to the proteasome by Ufd4, a homologous to E6-AP C-terminus (HECT)-class E3. Ubc7 is autoubiquitinated by a novel mechanism wherein the catalytic cysteine, instead of a lysine residue, provides the polyubiquitin chain acceptor site, and this cysteine-linked chain functions as a degradation signal. The polyubiquitin chain can also be transferred to a lysine side chain, suggesting a mechanism for polyubiquitin chain assembly that precedes substrate modification.

EGF Receptor Exposed to Oxidative Stress Acquires Abnormal Phosphorylation and Aberrant Activated Conformation That Impairs Canonical Dimerization

Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce “classical” receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.

NF-κB signaling: Flipping the Switch with Polyubiquitin Chains

Abstract Protein modification by ubiquitin has emerged as an important cellular regulatory mechanism. Recent studies illustrate the surprising ways in which polyubiquitin chains are manipulated in the regulation of NF-κB signaling.

Molecular Genetics of the Ubiquitin-Proteasome System : Lessons from Yeast

Our studies with the yeast Saccharomyces cerevisiae have uncovered a number of general principles governing substrate selectivity and proteolysis by the ubiquitin-proteasome system. The initial work focused on the degradation of a transcription factor, the MATalpha2 repressor, but the pathways uncovered have a much broader range of targets. At least two distinct ubiquitination mechanisms contribute to alpha2 turnover. One of them depends on a large integral membrane ubiquitin ligase (E3) and a pair of ubiquitin-conjugating enzymes (E2s). The transmembrane E3 and E2 proteins must travel from their site of synthesis in the ER to the inner nuclear membrane in order to reach nuclear substrates such as alpha2. The 26S proteasome is responsible for alpha2 degradation, and several important features of proteasome assembly and active site formation were uncovered. Most recently, we have delineated major steps in 20S proteasome assembly and have also identified several novel 20S proteasome assembly factors. Surprisingly, alterations in 20S proteasome assembly lead to defects in the assembly of the proteasome regulatory particle (RP). The RP associates with the 20S proteasome to form the 26S proteasome. Our data suggest that the 20S proteasome can function as an assembly factor for the RP, which would make it the first such factor for RP assembly identified to date.

Ceramide pathway and Apoptosis in Autoimmunity and Atherosclerosis

Publisher Summary The morphologic features of apoptosis are typical and well conserved in diverse cell types. This suggests a possible convergence of multiple signaling pathways, which ultimately culminate in one common route towards apoptosis. The ceramide/sphingomyelin (SM) pathway may constitute that common step. The studies have begun to identify a receptor-activated pathway of signal transduction involving the sphingolipid SM and its hydrolysis product ceramide. Apoptotic cell death was originally distinguished from necrosis based on morphological differences and on the tendency of necrotic, but not apoptotic, cells to induce an inflammatory response. Although it is still not clear how necrotic cells trigger inflammation, this capacity may be critical in relating tissue damage to the generation of an immune response. Apoptotic cell death, which concludes in the execution, packaging, and removal of the dying cells, has emerged as a physiological response to developmental and environmental signals. The pathways suggested for this significant process may vary between different types of cells.