Generation and physiological roles of linear ubiquitin chains

Ubiquitination now ranks with phosphorylation as one of the best-studied post-translational modifications of proteins with broad regulatory roles across all of biology. Ubiquitination usually involves the addition of ubiquitin chains to target protein molecules, and these may be of eight different types, seven of which involve the linkage of one of the seven internal lysine (K) residues in one ubiquitin molecule to the carboxy-terminal diglycine of the next. In the eighth, the so-called linear ubiquitin chains, the linkage is between the amino-terminal amino group of methionine on a ubiquitin that is conjugated with a target protein and the carboxy-terminal carboxy group of the incoming ubiquitin. Physiological roles are well established for K48-linked chains, which are essential for signaling proteasomal degradation of proteins, and for K63-linked chains, which play a part in recruitment of DNA repair enzymes, cell signaling and endocytosis. We focus here on linear ubiquitin chains, how they are assembled, and how three different avenues of research have indicated physiological roles for linear ubiquitination in innate and adaptive immunity and suppression of inflammation

Coordination chemistry and biology of chelators for the treatment of iron overload disorders

Treatment of the medical condition generally referred to as iron overload through the delivery of chelators has recently received a major boost. In 2005 Novartis gained FDA approval for the drug deferasirox, which may be taken orally. Until this time most patients with Fe overload have had to endure long periods of subcutaneous infusions of the orally ineffective drug desferrioxamine (desferal) which has led to major problems with patient compliance. An effective Fe chelator must possess a number of properties for it to be able to complex Fe in vivo and be excreted intact. This Perspective will provide an overview of the current state of chelators for Fe overload; both those currently approved and those undergoing preclinical development

Endothelial apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity

We recently cloned the full-length cDNA of a tumour-associated protein. The recombinant protein expressed in bacteria and referred to as angiocidin has potent antitumour activity in vivo and in vitro. Angiocidin inhibits tumour growth and angiogenesis by inducing apoptosis in endothelial cells. Based on the sequence similarity of angiocidin to S5a, one of the major polyubiquitin recognition proteins in eukaryotic cells, we postulated that the antiendothelial activity of angiocidin could be due in part to its polyubiquitin binding activity. In support of this hypothesis, we show that angiocidin binds polyubiquitin in vivo with high affinity and colocalises with ubiquitinated proteins on the surface of endothelial cells. Binding is blocked with an antiubiquitin antibody. Angiocidin treatment of endothelial cells transfected with a proteasome fluorescent reporter protein showed a dose-dependent inhibition of proteasome activity and accumulation of polyubiquitinated proteins. Full-length angiocidin bound polyubiquitin while three angiocidin recombinant proteins whose putative polyubiquitin binding sites were mutated either failed to bind polyubiquitin or had significantly diminished binding activity. The in vitro apoptotic activity of these mutants correlated with their polyubiquitin binding activity. These data strongly argue that the apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity

Identification of Genes Affecting the Toxicity of Anti-Cancer Drug Bortezomib by Genome-Wide Screening in S. pombe

Bortezomib/PS-341/Velcade, a proteasome inhibitor, is widely used to treat multiple myeloma. While several mechanisms of the cytotoxicity of the drug were proposed, the actual mechanism remains elusive. We aimed to identify genes affecting the cytotoxicity of Bortezomib in the fission yeast S.pombe as the drug inhibits this organism's cell division cycle like proteasome mutants. Among the 2815 genes screened (covering 56% of total ORFs), 19 genes, whose deletions induce strong synthetic lethality with Bortezomib, were identified. The products of the 19 genes included four ubiquitin enzymes and one nuclear proteasome factor, and 13 of them are conserved in humans. Our results will provide useful information for understanding the actions of Bortezomib within cells

ASPP: a new family of oncogenes and tumour suppressor genes

The apoptosis stimulating proteins of p53 (ASPP) family consists of three members, ASPP1, ASPP2 and iASPP. They bind to proteins that are key players in controlling apoptosis (p53, Bcl-2 and RelA/p65) and cell growth (APCL, PP1). So far, the best-known function of the ASPP family members is their ability to regulate the apoptotic function of p53 and its family members, p63 and p73. Biochemical and genetic evidence has shown that ASPP1 and ASPP2 activate, whereas iASPP inhibits, the apoptotic but not the cell-cycle arrest function of p53. The p53 tumour suppressor gene, one of the most frequently mutated genes in human cancer, is capable of suppressing tumour growth through its ability to induce apoptosis or cell-cycle arrest. Thus, the ASPP family of proteins helps to determine how cells choose to die and may therefore be a novel target for cancer therapy

Structural Organization of the 19S Proteasome Lid: Insights from MS of Intact Complexes

The 26S proteasome contains a 19S regulatory particle that selects and unfolds ubiquitinated substrates for degradation in the 20S catalytic particle. To date there are no high-resolution structures of the 19S assembly, nor of the lid or base subcomplexes that constitute the 19S. Mass spectra of the intact lid complex from Saccharomyces cerevisiae show that eight of the nine subunits are present stoichiometrically and that a stable tetrameric subcomplex forms in solution. Application of tandem mass spectrometry to the intact lid complex reveals the subunit architecture, while the coupling of a cross-linking approach identifies further interaction partners. Taking together our results with previous analyses we are able to construct a comprehensive interaction map. In summary, our findings allow us to identify a scaffold for the assembly of the particle and to propose a regulatory mechanism that prevents exposure of the active site until assembly is complete. More generally, the results highlight the potential of mass spectrometry to add crucial insight into the structural organization of an endogenous, wild-type complex

Degradation of Cdc25A by \u3b2-TrCP during S phase and in response to DNA damage

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A1\u20133. These events stimulate the ubiquitin-mediated pro- teolysis of Cdc25A1,4,5 and contribute to delaying cell-cycle progression, thereby preventing genomic instability1\u20137. Here we report that b-TrCP is the F-box protein that targets phosphory- lated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of b-TrCP1 and b-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that b-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of b-TrCP expression results in radioresistant DNA synthesis in response to DNA damage\u2014a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that b-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation

An Excellent Monitoring System for Surface Ubiquitination-Induced Internalization in Mammals

Background. At present, it is difficult to visualize the internalization of surface receptors induced by ubiquitination that is taken place at the plasma membrane in mammals. This problem makes it difficult to reveal molecular basis for ubiquitinationmediated internalization in mammals. Methodology/Principle Findings. In order to overcome it, we have generated T-REx-c-MIR, a novel mammalian Tet-on B cell line using a constitutively active E3 ubiquitin ligase, c-MIR, and its artificial target molecule. By applying the surface biotinylation method to T-REx-c-MIR, we succeeded to monitor the fate of surface target molecules after initiation of ubiquitination process by doxycycline (Dox)-induced c-MIR expression. Target molecules that preexisted at the plasma membrane before induction of c-MIR expression were oligo-ubiquitinated and degraded by Dox-induced c-MIR expression. Dox-induced c-MIR expression initiated rapid internalization of surface target molecules, and blockage of the internalization induced the accumulation of the surface target molecules that were newly ubiquitinated by c-MIR. Inhibition of the surface ubiquitination by down-regulating ubiquitin conjugating enzyme E2 impaired the internalization of target molecules. Finally, a complex of c-MIR and target molecule was detected at the plasma membrane. Conclusions/ Significances. These results demonstrate that in T-REx-c-MIR, surface target molecule is ubiquitinated at the plasma membrane and followed by being internalized from the plasma membrane. Thus, T-REx-c-MIR is a useful experimental tool t