Components Of Ubiquitination Pathway Research Articles

The Small Molecules Targeting Ubiquitin-Proteasome System for Cancer Therapy.

The ubiquitin-proteasome system (UPS) is the major pathway for degrading the intracellular proteins. The 2004 Nobel Prize in Chemistry was awarded to Rose, Hershko, and Ciechanover to highlight the fundamental importance of UPS. The alterations in this process have been shown to contribute to the cancer progression. Hence, UPS has become a popular target for developing chemotherapeutics against tumours. The application of bortezomib showed high efficiency in treating haematological malignancies by interfering with UPS activity. Many compounds are being screened and evaluated in recent pharmacological advances, either as single agents or in synergy with other drugs, and more to be revealed. In the present review, we exhibit the crucial ingredients involved in UPS and discuss the current situation of small molecules targeting various components of ubiquitination pathway in cancer treatment.

CacyBP/SIP promotes the proliferation of colon cancer cells

CacyBP/SIP is a component of the ubiquitin pathway and is overexpressed in several transformed tumor tissues, including colon cancer, which is one of the most common cancers worldwide. It is unknown whether CacyBP/SIP promotes the proliferation of colon cancer cells. This study examined the expression level, subcellular localization, and binding activity of CacyBP/SIP in human colon cancer cells in the presence and absence of the hormone gastrin. We found that CacyBP/SIP was expressed in a high percentage of colon cancer cells, but not in normal colonic surface epithelium. CacyBP/SIP promoted the cell proliferation of colon cancer cells under both basal and gastrin stimulated conditions as shown by knockdown studies. Gastrin stimulation triggered the translocation of CacyBP/SIP to the nucleus, and enhanced interaction between CacyBP/SIP and SKP1, a key component of ubiquitination pathway which further mediated the proteasome-dependent degradation of p27kip1 protein. The gastrin induced reduction in p27kip1 was prevented when cells were treated with the proteasome inhibitor MG132. These results suggest that CacyBP/SIP may be promoting growth of colon cancer cells by enhancing ubiquitin-mediated degradation of p27kip1.

Implicating SCF complexes in organogenesis in Caenorhabditis elegans.

Development of the Caenorhabditis elegans foregut (pharynx) is regulated by a network of proteins that includes the Retinoblastoma protein (pRb) ortholog LIN-35; the ubiquitin pathway components UBC-18 and ARI-1; and PHA-1, a cytoplasmic protein. Loss of pha-1 activity impairs pharyngeal development and body morphogenesis, leading to embryonic arrest. We have used a genetic suppressor approach to dissect this complex pathway. The lethality of pha-1 mutants is suppressed by loss-of-function mutations in sup-35/ztf-21 and sup-37/ztf-12, which encode Zn-finger proteins, and by mutations in sup-36. Here we show that sup-36 encodes a divergent Skp1 family member that binds to several F-box proteins and the microtubule-associated protein PLT-1/τ. Like SUP-35, SUP-36 levels were negatively regulated by UBC-18-ARI-1. We also found that SUP-35 and SUP-37 physically associated and that SUP-35 could bind microtubules. Thus, SUP-35, SUP-36, and SUP-37 may function within a pathway or complex that includes cytoskeletal components. Additionally, SUP-36 may regulate the subcellular localization of SUP-35 during embryogenesis. We carried out a genome-wide RNAi screen to identify additional regulators of this network and identified 39 genes, most of which are associated with transcriptional regulation. Twenty-three of these genes acted via the LIN-35 pathway. In addition, several S-phase kinase-associated protein (Skp)1-Cullin-F-Box (SCF) components were identified, further implicating SCF complexes as part of the greater network controlling pharyngeal development.

Giant virus with a remarkable complement of genes infects marine zooplankton

As major consumers of heterotrophic bacteria and phytoplankton, microzooplankton are a critical link in aquatic foodwebs. Here, we show that a major marine microflagellate grazer is infected by a giant virus, Cafeteria roenbergensis virus (CroV), which has the largest genome of any described marine virus (≈730 kb of double-stranded DNA). The central 618-kb coding part of this AT-rich genome contains 544 predicted protein-coding genes; putative early and late promoter motifs have been detected and assigned to 191 and 72 of them, respectively, and at least 274 genes were expressed during infection. The diverse coding potential of CroV includes predicted translation factors, DNA repair enzymes such as DNA mismatch repair protein MutS and two photolyases, multiple ubiquitin pathway components, four intein elements, and 22 tRNAs. Many genes including isoleucyl-tRNA synthetase, eIF-2γ, and an Elp3-like histone acetyltransferase are usually not found in viruses. We also discovered a 38-kb genomic region of putative bacterial origin, which encodes several predicted carbohydrate metabolizing enzymes, including an entire pathway for the biosynthesis of 3-deoxy-d-manno-octulosonate, a key component of the outer membrane in Gram-negative bacteria. Phylogenetic analysis indicates that CroV is a nucleocytoplasmic large DNA virus, with Acanthamoeba polyphaga mimivirus as its closest relative, although less than one-third of the genes of CroV have homologs in Mimivirus. CroV is a highly complex marine virus and the only virus studied in genetic detail that infects one of the major groups of predators in the oceans.

Homoserine Toxicity in Saccharomyces cerevisiae and Candida albicans Homoserine Kinase ( thr1 Δ) Mutants

In addition to threonine auxotrophy, mutation of the Saccharomyces cerevisiae threonine biosynthetic genes THR1 (encoding homoserine kinase) and THR4 (encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate homoserine. Moreover, homoserine is also toxic for Candida albicans thr1Delta mutants. Since increasing levels of threonine, but not other amino acids, overcome the homoserine toxicity of thr1Delta mutants, homoserine may act as a toxic threonine analog. Homoserine-mediated lethality of thr1Delta mutants is blocked by cycloheximide, consistent with a role for protein synthesis in this lethality. We identified various proteasome and ubiquitin pathway components that either when mutated or present in high copy numbers suppressed the thr1Delta mutant homoserine toxicity. Since the doa4Delta and proteasome mutants identified have reduced ubiquitin- and/or proteasome-mediated proteolysis, the degradation of a particular protein or subset of proteins likely contributes to homoserine toxicity.

The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells

Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation. UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27(Kip1) were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for beta-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells. UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27(Kip1), and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse. UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.

Exposure of Lemna minor to Arsenite: Expression Levels of the Components and Intermediates of the Ubiquitin/Proteasome Pathway

In animal cells, arsenite has been reported to cause sulfhydryl depletion, generate reactive oxygen species and increase the level of large ubiquitin-protein conjugates. Plant viability tests and DNA laddering experiments have shown that Lemna minor remains viable after exposure to 50 microM NaAsO(2) for periods of at least 6 h. However, protein metabolism is affected in two major ways: the synthesis of an array of stress proteins, which confer thermotolerance; and an increase in the amount of large ubiquitin-protein conjugates, particularly evident after 2-3 h of stress, indicative of a role for the ubiquitin/proteasome pathway. This outcome is primarily attributed to an increased availability of protein substrates during arsenite treatment for three main reasons: an increase in protein carbonyl content after 1-2 h of stress; moderate increments in the transcript levels of the sequences coding for the ubiquitin pathway components chosen as markers (polyubiquitin, E1 and E2, and the beta subunit and the ATPase subunits of the 26S proteasome); the observed increase in ubiquitin conjugates does not depend on de novo protein synthesis. This study is the first report on the involvement of the ubiquitin/proteasome pathway in response to arsenite in plants. In addition, it addresses the simultaneous expression of selected genes encoding the various components of the pathway. The results suggest that in plants, unlike in animals, the response to a relatively low level of arsenite does not induce apoptotic cell death. As a whole, the response to arsenite apparently involves a conjugation of salvage and proteolytic machineries, including heat shock protein synthesis and the ubiquitin/proteasome pathway.

An Integrated Mass Spectrometry-based Proteomic Approach

We developed an integrated proteomic approach to decipher in vivo protein-protein interactions and applied this strategy to globally map the 26 S proteasome interaction network in yeast. We termed this approach QTAX for quantitative analysis of tandem affinity purified in vivo cross-linked (X) protein complexes. For this work, in vivo formaldehyde cross-linking was used to freeze both stable and transient interactions occurring in intact cells prior to lysis. To isolate cross-linked protein complexes with high purification efficiency under fully denaturing conditions, a new tandem affinity tag consisting of a hexahistidine sequence and an in vivo biotinylation signal was adopted for affinity-based purification. Tandem affinity purification after in vivo cross-linking was combined with tandem mass spectrometry coupled with a quantitative SILAC (stable isotope labeling of amino acids in cell culture) strategy to carry out unambiguous protein identification and quantification of specific protein interactions. Using this method, we captured and identified the full composition of yeast 26 S proteasome complex as well as the two known ubiquitin receptors, Rad23 and Dsk2. Quantitative mass spectrometry analysis allowed us to distinguish specific proteasome-interacting proteins (PIPs) from background proteins and led to the identification of a total of 64 potential PIPs of which 42 are novel interactions. Among the 64 putative specific PIPs, there are ubiquitin pathway components, ubiquitinated substrates, chaperones, and transcription and translation regulators, demonstrating the efficacy of the developed approach in capturing in vivo protein interactions. The method offers an advanced technical approach to elucidate the dynamic protein interaction networks of the proteasome and can find a wide range of applications in the studies of other macromolecular protein complex interaction networks.

Ubiquitin-mediated pathways in C. elegans

Ubiquitin is a highly conserved 76 amino acid polypeptide, which is covalently attached to target proteins to signal their degradation by the 26S proteasome or to modify their function or localization. Regulated protein degradation, which is associated with many dynamic cellular processes, occurs predominantly via the ubiquitin-proteasome system. Ubiquitin is conjugated to target proteins through the sequential actions of a ubiquitin-activating enzyme, ubiquitin-conjugating enzymes, and ubiquitin-protein ligases. The nematode Caenorhabditis elegans has one ubiquitin-activating enzyme, twenty putative ubiquitin-conjugating enzymes, and potentially hundreds of ubiquitin-protein ligases. Research in C. elegans has focused on the cellular functions of ubiquitin pathway components in the context of organismal development. A combination of forward genetics, reverse genetics, and genome-wide RNAi screens has provided information on the loss-of-function phenotypes for the majority of C. elegans ubiquitin pathway components. Additionally, detailed analysis of several classes of ubiquitin-protein ligases has led to the identification of their substrates and the molecular pathways that they regulate. This review presents a comprehensive overview of ubiquitin-mediated pathways in C. elegans with a description of the known components and their identified molecular, cellular, and developmental functions.

Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway.

B-Myb, a highly conserved member of the Myb oncoprotein family, is a 110 kDa sequence-specific DNA binding protein expressed in virtually all proliferating cells. B-myb expression reaches its maximum at the G1/S phase boundary and during the S phase of the cell cycle. We have previously shown that B-Myb activity is cell cycle regulated and it is controlled by the antagonistic effects of cyclin D1 and A. Here we show that ectopic expression of cyclin A causes a pronounced reduction of B-Myb protein level. We provide evidence that in addition to triggering B-Myb activity an important effect of cyclin A is to facilitate multiple ubiquitination of B-Myb. The C-terminal domain of B-Myb is of key importance in mediating this effect of cyclin A. Contrary to full-length B-Myb, a C-terminal deletion mutant displays activity irrespective of cyclin A expression, does not undergo ubiquitination, and its half-life is not affected by cyclin A. Ectopic expression of either Cdc34 or the F-box protein p45Skp2, respectively the E2 and E3 components of a ubiquitination pathway that regulates the G1/S transition, accelerates degradation of B-Myb. We show that B-Myb physically and functionally interacts with components of the Cdc34-SCFp45Skp2 ubiquitin pathway and propose that B-Myb degradation may be required for controlling the correct alternation of events during progression through the cell division cycle. Oncogene (2000).

Ubiquitin-Dependent Pathway is Up-Regulated in Differentiating Lens Cells

The mammalian eye lens is composed of two distinct types of cells, epithelial cells and fiber cells. The fiber cells are generated throughout life via continuous differentiation of epithelial cells. Differentiation of lens cells involves dramatic changes in cellular components including altered activity of the ubiquitin dependent pathway. The concentration of high mass ubiquitin conjugates in the mitotically active-, differentiating-equatorial epithelial cells was 5–10 fold higher than that observed in mitotically quiescent, non-differentiated, central epithelial cells, even though there was a significant dilution of non-crystallin proteins due to an increase in level of crystallins in the differentiating cells. Similar observations were made when differentiation was modeled by exposure of lens epithelial explants to bFGF in culture. Activities of ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) in the differentiating equatorial epithelial cells were also up to 100% higher than those noted in non-differentiated central epithelial cells and E1 appears to be rate controlling for ubiquitinylation. Consistent with the higher concentrations of high mass ubiquitin conjugates, there was a trend of enhanced ability to execute ATP-dependent protein degradation in the differentiating equatorial epithelial cells as compared with degradation in the non-differentiated central epithelial cells. These data indicate that the ubiquitin dependent pathway is up-regulated during differentiation of lens cells. In the differentiated fibers, the concentration of high mass ubiquitin conjugates and relative activities of E1 and E2s were 50% lower than in the non-differentiated central epithelial cells. In comparison, the concentration of the 110 kDa E1 was unchanged in differentiated fibers. However, if the factor of dilution by the significant increase in the level of crystallins was taken into account, the level or activities of the components of ubiquitin pathway in the differentiated cells was higher than the level noted in non-differentiated cells. These data indicate that, as compared with other non-crystallin proteins, there is differential stabilization and/or synthesis of the 110 kDa E1 and some other components of the ubiquitin dependent pathway in differentiated fibers.