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Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC , UBB and RPS27A , in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1 , HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker β-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.

Ubiquitin (Ub) is a small 76-amino acid protein that is engaged in many different pathways within the cell, including protein turnover. During proteotoxic stress, when the demand of clearing damaged/misfolded proteins strongly increases, cells activate Ub gene transcription to face the need of extra ubiquitin. This paper shows the contribution of the four ubiquitin coding genes (UBB, UBC, UBA52, RPS27A) to the ubiquitin RNA pool under basal and stressful conditions. Our results reveal that UBC and RPS27A represent the major fraction of the Ub transcriptome in different cell lines, but when converted to the coding potential, polyubiquitin genes UBC and UBB mainly contribute to determine the intracellular ubiquitin content under basal conditions. Both the polyubiquitin genes UBB and UBC are upregulated upon proteasome inhibition and oxidative stress, with markedly higher responses from the UBC promoter. A similar output, with lower fold-inductions, is detected in heat-stressed cells, with UBC acting as the main contributor to thermotolerance. By contrast, upon these stressors, the levels of UBA52 and RPS27A mRNAs remain unchanged. Remarkably, UV irradiation fails to induce Ub gene transcription, but rather seems to act at the post-transcriptional level, by stabilizing ubiquitin mRNAs at UV doses which induce rapid degradation of other RNA molecules. Moreover, the evidence that the UBC core promoter contains multiple transcription start sites and their responsiveness to stress, is here reported for the first time., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The role of ubiquitylation in signal-induced activation of nuclear factor -kappaB (NF-kappaB) has been well established, while its involvement in maintaining NF-kappaB basal activity is less certain. Recent evidences demonstrate that in unstimulated cells, NF-kappaB homeostasis is actually the result of opposing forces: pro-activating activity of the IkappaB Kinase (IKK) and inhibitory activity of the Inhibitor of -kappaB (IkappaB) proteins. It is well known that endogenous de-ubiquitylating mechanisms are less effective on Ub motifs containing UbG76A. Here, we show that overexpression of a ubiquitin (Ub) G76A mutant leads to persistent activation of the IKK/NF-kappaB pathway in the absence of extra-cellular stimuli. In contrast, no effects on NF-kappaB activation were observed upon expression of UbK48R and UbK63R mutants, which are known to impair elongation of Lys(48)- and Lys(63)-linked poly-ubiquitin chains, respectively. Overall, these findings indicate that under basal conditions, the rate of de-ubiquitylation, rather than that of substrate ubiquitylation, is critical for the maintenance of appropriate levels of IKK/NF-kappaB activity.

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation.

The polyubiquitin gene ubiquitin C (UBC) is considered a stress protective gene and is upregulated under various stressful conditions, which is probably a consequence of an increased demand for ubiquitin in order to remove toxic misfolded proteins. We previously identified heat shock elements (HSEs) within the UBC promoter, which are responsible for heat shock factor (HSF)1‐driven induction of the UBC gene and are activated by proteotoxic stress. Here, we determined the molecular players driving the UBC gene transcriptional response to arsenite treatment, mainly addressing the role of the nuclear factor‐erythroid 2‐related factor 2 (Nrf2)‐mediated antioxidant pathway. Exposure of HeLa cells to arsenite caused a time‐dependent increase of UBC mRNA, while cell viability and proteasome activity were not affected. Nuclear accumulation of HSF1 and Nrf2 transcription factors was detected upon both arsenite and MG132 treatment, while HSF2 nuclear levels increased in MG132‐treated cells. Notably, siRNA‐mediated knockdown of Nrf2 did not reduce UBC transcription under either basal or stressful conditions, but significantly impaired the constitutive and inducible expression of well‐known antioxidant response element‐dependent genes. A chromatin immunoprecipitation assay consistently failed to detect Nrf2 binding to the UBC promoter sequence. By contrast, depletion of HSF1, but not HSF2, significantly compromised stress‐induced UBC expression. Critically, HSF1‐mediated UBC trans‐activation upon arsenite exposure relies on transcription factor binding to previously mapped distal HSEs, as demonstrated to occur under proteasome inhibition. These data highlight HSF1 as the pivotal transcription factor that translates different stress signals into UBC gene transcriptional induction. [ABSTRACT FROM AUTHOR]

The promoter of the polyubiquitin C gene (UBC) contains putative heat shock elements (HSEs) which are thought to mediate UBC induction upon stress. However, the mapping and the functional characterization of the cis-acting determinants for its up-regulation have not yet been addressed. In this study, the sequence encompassing 916 nucleotides upstream of the transcription start site of the human UBC gene has been dissected by in silico, in vitro and in vivo approaches. The information derived from this analysis was used to study the functional role and the interplay of the identified HSEs in mediating the transcriptional activation of the UBC gene under conditions of proteotoxic stress, induced by the proteasome inhibitor MG132. Here we demonstrate that at least three HSEs, with different configurations, exist in the UBC promoter: two distal, residing within nucleotides -841/-817 and -715/-691, and one proximal to the transcription start site (nt -100/-65). All of them are bound by transcription factors belonging to the heat shock factor (HSF) family, as determined by bandshift, supershift and ChIP analyses. Site-directed mutagenesis of reporter constructs demonstrated that while the distal elements are involved in the up-regulation of UBC in response to proteasome inhibition, the proximal one appears rather to function as negative regulator of the stress-induced transcriptional activity. This is the first evidence that an HSE may exert a negative role on the transcription driven by other HSE motifs on the same gene promoter, highlighting a new level of complexity in the regulation of HSFs and in the control of ubiquitin levels. [ABSTRACT FROM AUTHOR]

In a number of organisms, introns affect expression of the gene in which they are contained. Our previous studies revealed that the 5′-UTR intron of human ubiquitin C (UbC) gene is responsible for the boost of reporter gene expression and is able to bind, in vitro, Yin Yang 1 (YY1) trans-acting factor. In this work, we demonstrate that intact YY1 binding sequences are required for maximal promoter activity and YY1 silencing causes downregulation of luciferase mRNA levels. However, YY1 motifs fail to enhance gene expression when the intron is moved upstream of the proximal promoter, excluding the typical enhancer hypothesis and supporting a context-dependent action, like intron-mediated enhancement (IME). Yet, almost no expression is seen in the construct containing an unspliceable version of UbC intron, indicating that splicing is essential for promoter activity. Moreover, mutagenesis of YY1 binding sites and YY1 knockdown negatively affect UbC intron removal from both endogenous and reporter transcripts. Modulation of splicing efficiency by YY1 cis-elements and protein factor may thus be part of the mechanism(s) by which YY1 controls UbC promoter activity. Our data highlight the first evidence of the involvement of a sequence-specific DNA binding factor in IME. [ABSTRACT FROM AUTHOR]

Examines the ubiquitinated repeats in human erythroid α-spectrin. Importance of deformability and resistance to mechanical stress by the red cells; Susceptibility of the ubiquitination; Effects of mutation on the interaction resulting in the increase of membrane fragility.

Demonstrates the effects of the activation of the ubiquitin proteolytic system on IkappaBalpha in the lymph nodes of animals infected with murine acquired immunodeficiency syndrome. Proteosomal activity; Reduction in the level of IkappaBalpga in lymph nodes of infected mice; Absence of effect of IkappaBalpha degradation on nuclear factor-kappaB DNA binding activity.

Examines the alteration of α-spectrin ubiquitination due to age-dependent changes in erythrocyte membrane. Decrease of cell age-dependence; Dependence of ubiquitination on the source of the red cell membranes; Significance of membrane architecture for the decrease of age-dependent in ubiquitination.

K48R ubiquitin (K48R-Ub) is an analogue of native ubiquitin that does not form polyubiquitin chain conjugates. Targeted delivery of this recombinant mutant ubiquitin to human macrophages results in an intracellular increase in the ubiquitin analogue. IkBα polyubiquitination and degradation were significantly inhibited in K48R-Ub targeted macrophages upon stimulation with lipopolysaccharide. The ability to reduce IkBα degradation was also associated with a reduced production of TNF-α, the gene of which is under NF-kB control. At a concentration of 0.1 μM, dexamethasone was less effective than K48R-Ub in preventing IkBα depletion and TNF-α release. These data suggest that ubiquitin analogues are potent suppressors of TNF-α release in macrophages. [ABSTRACT FROM AUTHOR]

Demonstrates that in murine AIDS, the adenosine triphosphate-dependent and ubiquitin-dependent proteolytic system is strongly affected at least in the lymph nodes of infected mice. Features of retrovirus-induced murine AIDS including lymphoproliferation, chronic B-cell activation resulting in hypergammaglobulinemia and profound immunodeficiency; Coincidence of the accumulation of ubiquitin conjugates and induction of the conjugating system with an increase in the proteolytic activity.

Ubiquitin is a small protein involved in an ATP-dependent proteolytic pathway in all eukaryotes. This pathway has been demonstrated to be required for both the bulk degradation of cellular proteins and the targeted proteolysis of specific regulatory proteins. We have investigated the presence of ubiquitin (Ub) and the ubiquitin-conjugating system in dormant and activated tubers of Helianthus tuberosus L. cv. OB1 that represent a widely used model system for studies on the cell cycle in plants. Immunoblot experiments revealed the presence of free ubiquitin and ubiquitin conjugates. Furthermore, the presence of an active ubiquitin-conjugating system, both time- and ATP-dependent, was demonstrated by incubation with 125I-labeled ubiquitin. A few proteins able to form thiol esters with 125I-Ub and probably corresponding to ubiquitin-conjugating enzymes, El and E2s, were also found. During the first cell cycle, several proteins become ubiquitinated. In particular a large amount of protein conjugates was present at 6 h when the lowest content of free ubiquitin was found. Subsequently, a dramatic decrease in ubiquitin conjugates occurred. It is well known that cell cycle progression in eukaryotes depends on cyclin levels and cyclin B degradation is ubiquitin- and ATP-dependent. By immunoblot experiments we showed that cyclin B in H. tuberosus is present as at least two protein bands of 50 and 54 kDa and that their amounts undergo profound changes during the cell cycle. The 54-kDa band was also recognized by an anti-ubiquitin antibody. These data seem to indicate that in H. tuberosus activated tuber slices, the ATP-dependent ubiquitin proteolytic pathway is involved in the dedifferentiation process occurring after the artificial break of dormancy when the cells acquire the characteristics linked to the meristematic state. [ABSTRACT FROM AUTHOR]

Focuses on the use of engineered green fluorescent proteins as substrates for the quantification of the ubiquitin/proteasome-dependent proteolytic system in living cells. Effect of green fluorescent protein on mice; Limitations of the green fluorescent proteins.

Abstract: Ubiquitin (Ub) plays a crucial role in almost every aspect of cellular functions. It is encoded by four genes, of which UbC is known to meet cell demand for ubiquitin in both basal and stressful conditions. To understand the molecular mechanisms regulating UbC gene expression, we performed a functional characterization of the UbC promoter. Deletion analyses on the 5′ end of the −916/+878 promoter region, excluded the functional importance of nt −916/−371 in the transcriptional regulation of the gene, while 3′ deletions revealed that intron removal (nt+65/+876) resulted in a marked reduction of promoter activity in all the reporter constructs, regardless of the cell types. Intron substitution with a heterologous chimeric intron failed to restore promoter activity, thus allowing to exclude that the splicing event, per se, can be responsible for the intron-mediated burst of transcription. Gel shift assays demonstrated nuclear factor binding with the +137/+766 intron region. Reporter constructs carrying partial intron deletions confirmed that this sequence is, indeed, required for maximal transcriptional activity. Computer-based analysis found potential Sp1 binding motifs within the intron sequence and electrophoretic mobility shift and supershift assays demonstrated that both Sp1 and Sp3 transcription factors interact, in vitro, with the UbC intron, at multiple binding sites. Moreover, ectopic expression of Sp1 and Sp3 revealed that both transcription factors positively regulate UbC promoter activity. Collectively, our data highlight the very new evidence that the 5′-UTR intron is crucial in regulating UbC gene expression and provide insights into the pivotal role of Sp1/Sp3 binding to the intronic enhancer in the regulation of UbC transcription. [Copyright &y& Elsevier]