Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth.

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2(E301K)) suppressed the ubi4Δ growth defect. The rpt2(E301K) mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2(E301K) mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells.

Myofibrillar protein synthesis in myostatin-deficient mice.

Either increased protein synthesis or prolonged protein half-life is necessary to support the excessive muscle growth and maintenance of enlarged muscles in myostatin-deficient mice. This issue was addressed by determining in vivo rates of myofibrillar protein synthesis in mice with constitutive myostatin deficiency (Mstn(DeltaE3/DeltaE3)) or normal myostatin expression (Mstn(+/+)) by measuring tracer incorporation after a systemic flooding dose of l-[ring-(2)H(5)]phenylalanine. At 5-6 wk of age, Mstn(DeltaE3/DeltaE3) mice had increased muscle mass (40%), fractional rates of myofibrillar synthesis (14%), and protein synthesis per whole muscle (60%) relative to Mstn(+/+) mice. With maturation, fractional rates of synthesis declined >50% in parallel with decreased DNA and RNA [total, 28S rRNA, and poly(A) RNA] concentrations in muscle. At 6 mo of age, Mstn(DeltaE3/DeltaE3) mice had even greater increases in muscle mass (90%) and myofibrillar synthesis per muscle (85%) relative to Mstn(+/+) mice, but the fractional rate of synthesis was normal. Estimated myofibrillar protein half-life was not affected by myostatin deficiency. Muscle DNA concentrations were reduced in both young and mature Mstn(DeltaE3/DeltaE3) mice, whereas RNA concentrations were normal, so the ratio of RNA to DNA was approximately 30% greater than normal in Mstn(DeltaE3/DeltaE3) mice. Thus the increased protein synthesis and RNA content per muscle in myostatin-deficient mice cannot be explained entirely by an increased number of myonuclei.

Peroxisome proliferator-activated receptor beta (delta)-dependent regulation of ubiquitin C expression contributes to attenuation of skin carcinogenesis.

The role of peroxisome proliferator-activated receptor-beta (PPARbeta) in the molecular regulation of skin carcinogenesis was examined. Increased caspase-3 activity associated with apoptosis was found in the skin of wild-type mice after tumor promotion with 12-O-tetradecanoylphorbol-13-acetate, and this effect was diminished in PPARbeta-null mice. The onset of tumor formation, tumor size, and tumor multiplicity induced from a two-stage carcinogen bioassay (7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate) were significantly enhanced in PPARbeta-null mice compared with wild-type mice. To begin to characterize the molecular changes underlying this PPARbeta-dependent phenotype, microarray analysis was performed and a number of differentially regulated gene products were identified including ubiquitin C. Subsequent promoter analysis, reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays provide evidence that PPARbeta regulates ubiquitin C expression, and that ubiquitination of proteins is influenced by PPARbeta. These results strongly suggest that activation of PPARbeta-dependent target genes provides a novel strategy to inhibit tumor promotion and carcinogenesis.

Depletion of polyubiquitin encoded by the UBI4 gene confers pleiotropic phenotype to Candida albicans cells.

We have studied the roles of polyubiquitin in Candida albicans physiology. Heterologous expression of the C. albicans polyubiquitin (UBI4) gene in a ubi4 Saccharomyces cerevisiae strain suppressed the mutant phenotype (hypersensitivity to heat shock). A heterozygous strain UBI4/Deltaubi4::hisG, obtained following the ura-blaster procedure, was used to construct a conditional mutant using a pCaDis derivative plasmid. By serendipity we isolated the UBI4 conditional mutant as well as a UBI4 mutant containing a non-functional MET3 promoter. Depletion of polyubiquitin conferred pleiotropic effects to mutant cells: (i) a limited increased sensitivity to mild heat shock; (ii) increased formation of colony morphology variants; and (iii) induction of hyphal and pseudohypal development. These results indicate that polyubiquitin in C. albicans is involved in the negative control of switching, as well as in maintaining the yeast cell morphology, probably by silencing mechanisms triggering the hyphal and pseudohyphal development in the absence of environmental inducers.