p53 protein turnover through the ubiquitination pathway is a vital mechanism in the regulation of its transcriptional activity; however, little is known about p53 turnover through proteasome-independent pathway(s). The digestive organ expansion factor (Def) protein is essential for the development of digestive organs. In zebrafish, loss of function of def selectively upregulates the expression of p53 response genes, which raises a question as to what is the relationship between Def and p53. We report here that Def is a nucleolar protein and that loss of function of def leads to the upregulation of p53 protein, which surprisingly accumulates in the nucleoli. Our extensive studies have demonstrated that Def can mediate the degradation of p53 protein and that this process is independent of the proteasome pathway, but dependent on the activity of Calpain3, a cysteine protease. Our findings define a novel nucleolar pathway that regulates the turnover function of p53, which will advance our understanding of p53's role in organogenesis and tumorigenesis.

The molecular weight (MW) of a protein can be predicted based on its amino acids (AA) composition. However, in many cases a non-chemically modified protein shows an SDS PAGE-displayed MW larger than its predicted size. Some reports linked this fact to high content of acidic AA in the protein. However, the exact relationship between the acidic AA composition and the SDS PAGE-displayed MW is not established. Zebrafish nucleolar protein Def is composed of 753 AA and shows an SDS PAGE-displayed MW approximately 13 kDa larger than its predicted MW. The first 188 AA in Def is defined by a glutamate-rich region containing ~35.6% of acidic AA. In this report, we analyzed the relationship between the SDS PAGE-displayed MW of thirteen peptides derived from Def and the AA composition in each peptide. We found that the difference between the predicted and SDS PAGE-displayed MW showed a linear correlation with the percentage of acidic AA that fits the equation y = 276.5x − 31.33 (x represents the percentage of acidic AA, 11.4% ≤ x ≤ 51.1%; y represents the average ΔMW per AA). We demonstrated that this equation could be applied to predict the SDS PAGE-displayed MW for thirteen different natural acidic proteins.

Determining how plasticity of developmental traits responds to environmental conditions is a challenge that must combine evolutionary sciences, ecology, and developmental biology. During metamorphosis, fish alter their morphology and color pattern according to environmental cues. We observed that juvenile clownfish (Amphiprion percula) modulate the developmental timing of their adult white bar formation during metamorphosis depending on the sea anemone species in which they are recruited. We observed an earlier formation of white bars when clownfish developed with Stichodactyla gigantea (Sg) than with Heteractis magnifica (Hm). As these bars, composed of iridophores, form during metamorphosis, we hypothesized that timing of their development may be thyroid hormone (TH) dependent. We treated clownfish larvae with TH and found that white bars developed earlier than in control fish. We further observed higher TH levels, associated with rapid white bar formation, in juveniles recruited in Sg than in Hm, explaining the faster white bar formation. Transcriptomic analysis of Sg recruits revealed higher expression of duox, a dual oxidase implicated in TH production as compared to Hm recruits. Finally, we showed that duox is an essential regulator of iridophore pattern timing in zebrafish. Taken together, our results suggest that TH controls the timing of adult color pattern formation and that shifts in duox expression and TH levels are associated with ecological differences resulting in divergent ontogenetic trajectories in color pattern development.

Expression of the lactate dehydrogenase A subunit (LDH-A) gene can be controlled by transcriptional as well as posttranscriptional mechanisms. In rat C6 glioma cells, LDH-A mRNA is stabilized by activation and synergistic interaction of protein kinases A and C. In the present study, we aimed to identify the sequence domain which determines and regulates mRNA stability/ instability by protein kinase A and focused our attention on the 3-untranslated region (3-UTR) of LDH-A mRNA. We have constructed various chimeric globin/ lactate dehydrogenase (ldh) genes linked to the c-fos promoter and stably transfected them into rat C6 glioma cells. After their transfection, we determined the halflife of transcribed chimeric globin/ldh mRNAs. The results showed that at least three sequence domains within the LDH- Analysis of the LDH 1 isoenzyme patterns in various cell types under a variety of physiologic conditions suggests complex regulatory mechanisms that determine specific isoenzyme expression (1-7). The LDH-A subunit, for instance, is subject to regulation by a number of different effector agents such as estrogen (3,8), epidermal growth factor (5), catecholamines (4, 9), phorbol ester (7), and c-Myc (10), which change the isoenzyme pattern almost exclusively in favor of the LDH-5 (A4) isoenzyme. The functional importance of these LDH isoenzyme shifts is generally attributed to a need for increased A subunitcontaining isoforms (such as LDH-4 or -5), which can derive more energy from the anaerobic pathway by reducing pyruvate to lactate. Investigations into the mechanism of LDH-A gene expression has identified two basic controls consisting of a transcription-regulatory cascade (4, 6, 11) and a mechanism that regulates the half-life of LDH-A mRNA (4, 12), both of which are major determinants of intracellular LDH-A mRNA levels.Messenger RNA turnover rates fluctuate over a wide range, and it is important to identify and characterize putative stability-regulating mRNA domains and their interacting factors that may be responsible for these functional effects. A great number of reports have demonstrated the existence of such domains and their trans-acting regulatory factors that are critical in determining the half-life of mRNA (13). Several of these studies indicate that the stability of some, but not all, mRNA is determined by specific cis-acting AU-rich domains located in the 3Ј-UTR. For example, a number of mRNAs such as cytokine, lymphokine and protooncogene mRNAs share a common sequence motif with a high content of A and U nucleotides in the 3Ј-UTR (14) and exhibit half-lives in the range of only a fraction of 1 h (15-18). In addition, attention has focused on modulation of mRNA stability in response to a variety of physiological signals. For instance, histone mRNA stability is regulated by the cell cycle (19) and intracellular iron levels control the stability of transferrin receptor mRNA (20,21). Moreover, manipulation of cells with several different effector agents can alter the steady-state level of mRNA during cell g...

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.