Disruption of the polyubiquitin gene Ubb causes retinal degeneration in mice.

We have previously demonstrated that a reduction in ubiquitin (Ub) levels via disruption of the polyubiquitin gene Ubb results in reactive gliosis and hypothalamic neurodegeneration in mice. However, it is not known whether other neural tissues, apart from the brain, can also be affected by Ubb disruption. We examined the retina, which, being derived from the diencephalon, has the same developmental origin as the hypothalamus. We found that expression levels of Ubb were much higher than those of the other polyubiquitin gene Ubc in the retina. In retinal tissues from Ubb knockout (KO) mice, we found that Ubc expression was upregulated to compensate for the loss of Ubb; however, the Ub pool remained disrupted, with reduced levels of free Ub. To directly demonstrate whether the disrupted Ub pools affect neural integrity in retinal tissues, we investigated retinal layers in control and Ubb KO mice. Using optical coherence tomography and histological analysis, we demonstrated that the thickness of the outer nuclear layer of the retina was decreased in Ubb KO mice compared to control mice, suggesting that retinal degeneration was induced by Ub deficiency. Furthermore, the mRNA and protein levels of rhodopsin decreased and those of glial fibrillary acidic protein increased in Ubb KO mouse retinas. Therefore, the maintenance of Ub pools in the retina appears to be crucial for the survival of photoreceptor cells and the prevention of excessive glial cell activation.

A fluorescence polarization-based competition assay for measuring interactions between unlabeled ubiquitin chains and UCH37•RPN13.

Ubiquitin chains regulate distinct signaling events through cooperative interactions with effector proteins and deubiquitinases. Measuring the strength of these interactions is often challenging; either large amounts of material are required or one of the binding partners must be labeled for detection. We sought to develop a label-free method for measuring binding of ubiquitin chains to the proteasome-associated deubiquitinase UCH37 and its binding partner RPN13. The method we describe here is based on a fluorescence polarization competition (FPcomp) assay in which fluorescent monoubiquitin is competed off the UCH37•RPN13 complex by the addition of unlabeled ubiquitin chains. We show that the UCH37•RPN13 complex displays higher affinity toward chains with more than two ubiquitin subunits. Removing the ubiquitin-binding PRU domain of RPN13 does not change affinities. These results suggest UCH37•RPN13 acts to selectively recruit proteins modified with long chains (>2 subunits) to the proteasome for degradation. We also demonstrate that the FPcomp assay is suitable for high-throughput screening, which is important considering both UCH37 and RPN13 are potential targets for cancer therapy.

Ub-ProT reveals global length and composition of protein ubiquitylation in cells.

Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.

Metabolite extract of Streptomyces hygroscopicus Hygroscopicus inhibit the growth of Plasmodium berghei through inhibition of ubiquitin - proteasome system.

Streptomyces hygroscopicus Hygroscopicus, a member of family of Actinomycetes produces eponemycin a proteasome inhibitor that can inhibit Ubiquitin-Proteasome System (UPS) function in eukaryotic cell. Previous study showed that coronamycin, an active substrate isolated from Streptomyces sp. can act as anti-plasmodial, antibacterial, and antifungal, however the research did not show the mechanism of coronamycin in inhibiting the growth of Plasmodium. This research was done to reveal if eponemycin that is contained in metabolite extract of S. hygroscopicus can inhibit UPS function of Plasmodium berghei. This study was an experimental study using P. berghei infected Balb/C mice as malaria model. Samples were divided into 1 control group (group infected with P. berghei without treatment) and 3 treatment groups (mice infected with P. berghei and treated intra-peritoneal with metabolite extract of S. hygroscopicus dose 130 µg/kgBW, 580 µg/kgBW, and 2600 µg/kgBW for 5 days). The degree of parasitemia and morphology of the parasite were measured from the first day of malaria induction until the last treatment. The accumulation level of polyubiquitin was measured using Western blot and ELISA method. The degree of parasitemia on day 6 showed significant differences among treatment groups and control (p=0,000). Percentage of inhibition showed significant differences between control and group treated with metabolite extract of S. hygroscopicus 2600 µg/kgBW. An increasing dose of extract of S. hygroscopicus followed by an increasing of inhibition in parasite growth (r=0,850). Probit analysis showed that ED50 was 9.418 µg/kgBW. There was a change in morphology of the parasite after treatment. Parasite morphology became crisis form. There was an accumulation of polyubiquitinated protein in the group treated with metabolite extract of S. hygroscopicus 2600 µg/kgBW. It can be concluded that analog eponemycin in metabolite of S. hygroscopicus is a potential candidate for new malarial drug by inhibiting UPS function of the parasite and cause stress and dead of the parasite.

Synthesis and analysis of linear ubiquitin chains.

Previously, polyubiquitin chains have been believed to be generated through isopeptide linkages between C-terminal of carboxyl group of ubiquitin and ε-amino group of one of the seven lysine residues in another ubiquitin. In 2006, a new type of polyubiquitin chain was identified in which the C-terminal carboxyl group of one ubiquitin is conjugated to α-amino group of the N-terminal methionine of another ubiquitin. The new type of polyubiquitin was named as the linear polyubiquitin chain. Linear polyubiquitin chains were shown to be involved in NF-κB activation. Here, we describe methods to synthesize linear polyubiquitin chains in vitro and to detect linear chains in vivo.

Using native gel electrophoresis and phosphofluoroimaging to analyze GFP-tagged proteasomes.

Native polyacrylamide gel electrophoresis (PAGE) is an invaluable technique in biochemistry to characterize native protein complexes with high molecular mass. Thus, native PAGE is suited to resolve proteasomes, giant proteases responsible for the degradation of polyubiquitylated proteins. Proteasomes contain multiple subunits and exist in different configurations. All configurations have a common 20S core particle (CP). The CP encloses the proteolytic chamber and is composed of four stacked rings with C2 symmetry. The entrance to the CP is gated by central pores within the outer rings, which also provide the binding sites for the 19S regulatory complex (RP). Adjacent regulatory proteins, such as Blm10/PA200, are bound to specific proteasome species of low abundance and contribute to the heterogeneity of proteasome complexes. To get insight into the complexity of proteasome configurations in yeast, we developed a native PAGE system by which GFP-labelled variants of proteasomal subunits are visualized by phosphofluoroimaging. Following native PAGE, proteasome species can be subjected to in-gel activity assays, subsequent SDS-PAGE, and Western blotting.

Arabidopsis thaliana 26S proteasome subunits RPT2a and RPT5a are crucial for zinc deficiency-tolerance.

RPTs (regulatory particle triple-A-ATPase) are components of 26S proteasome. We found novel roles of RPT2a and RPT5a in Zn deficiency-tolerance. Arabidopsis thaliana mutants carrying T-DNA in RPT2a and RPT5a were more sensitive to Zn deficiency than the wild-type. In the rpt mutants, the shoot Zn contents were similar to those of the wild-type. Transcripts of Zn deficiency-inducible genes were highly accumulated in the rpt mutants, suggesting that the rpt mutants suffer from various Zn deficiency symptoms, although the Zn levels are not reduced. Lipid peroxidation levels, known to be increased under Zn deficiency, were higher in the rpt mutants than in the wild-type. Poly-ubiquitinated proteins were accumulated upon exposure to Zn deficiency, especially in the rpt mutants. Overall, this study indicates that RPT2a and RPT5a are involved in Zn deficiency-tolerance, possibly through alleviation of oxidative stresses and/or processing of poly-ubiquitinated proteins.

Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes.

The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis.

BACKGROUND: Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection. RESULTS: Using wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG-tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity. CONCLUSION: In this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin.

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

Molt cycle-dependent molecular chaperone and polyubiquitin gene expression in lobster.

Lobster claw muscle undergoes atrophy in correlation with increasing ecdysteroid (steroid molting hormone) titers during premolt. In vivo molecular chaperone (constitutive heat shock protein 70 [Hsc70], heat shock protein 70 [Hsp70], and Hsp90) and polyubiquitin messenger ribonucleic acid (mRNA) levels were examined in claw and abdominal muscles from individual premolt or intermolt lobsters. Polyubiquitin gene expression was assayed as a marker for muscle atrophy. Both Hsc70 and Hsp90 mRNA levels were significantly induced in premolt relative to intermolt lobster claw muscle, whereas Hsp70 mRNA levels were not. Hsp90 gene expression was significantly higher in premolt claw muscle when compared with abdominal muscle. Polyubiquitin mRNA levels were elevated in premolt when compared with intermolt claw muscle and significantly elevated relative to premolt abdominal muscle.