Kristin Breitschopf

Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.

The epithelial Na+ channel (ENaC), composed of three subunits (αβγ), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin‐protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short‐lived protein (t1/2 ∼1 h) that is ubiquitinated in vivo on the α and γ (but not β) subunits. Mutation of a cluster of Lys residues (to Arg) at the N‐terminus of γENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N‐terminal Lys to Arg mutations in αENaC, but not in βENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell‐surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled αβγENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination‐mediated regulation of ion channels.

Nitric oxide inhibits caspase-3 by S-nitrosation in vivo.

In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. In vitro data have demonstrated that NO inhibits the activity of caspase-3 byS-nitrosation of the enzyme. Here we present evidence for the in vivo occurrence and functional relevance of this novel antiapoptotic mechanism. To demonstrate that the cysteine residue Cys-163 of caspase-3 is S-nitrosated, cells were transfected with the Myc-tagged p17 subunit of caspase-3. After incubation of the transfected cells with different NO donors, Myc-tagged p17 was immunoprecipitated with anti-Myc antibody.S-Nitrosothiol was detected in the immunoprecipitate by electron spin resonance spectroscopy after liberation and spin trapping of NO byN-methyl-d-glucamine-dithiocarbamate-iron complex. Transfection of cells with a p17 mutant, where the essential Cys-163 was mutated into alanine, completely preventedS-nitrosation of the enzyme. As a functional correlate, in human umbilical vein endothelial cells the NO donors sodium nitroprusside or PAPA NONOate (50 μm) significantly reduced the increase in caspase-3-like activity induced by overexpressing caspase-3 by 75 and 70%, respectively. When human umbilical vein endothelial cells were cotransfected with β-galactosidase, morphological analysis of stained cells revealed that cell death induction by overexpression of caspase-3 was completely suppressed in the presence of sodium nitroprusside, PAPA NONOate, or S-nitroso-l-cysteine (50 μm). Thus, NO supplied by exogenous NO donors serves in vivo as an antiapoptotic regulator of caspase activity viaS-nitrosation of the Cys-163 residue of caspase-3.

Posttranslational Modification of Bcl-2 Facilitates Its Proteasome-Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway

ABSTRACT The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-α) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. We further demonstrated that incubation with TNF-α induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative stress mediates TNF-α-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells.

Nitric Oxide Activates Telomerase and Delays Endothelial Cell Senescence

Endothelial cells (ECs) undergo a limited number of cell divisions, ultimately stop dividing, and enter a state that is designated replicative senescence. Shortening of telomeres is believed to be a molecular clock that triggers senescence. Telomerase, a RNA-directed DNA polymerase, extends telomeres of eukaryotic chromosomes and delays the development of senescence. In this study, we examined telomere length and the activity of telomerase during aging of human ECs in culture and elucidated the effect of nitric oxide (NO). A significant increase in senescent cells as detected by acidic &bgr;-galactosidase expression and a reduction of telomere length were found after 11 passages. Telomerase activity was reduced after the seventh passage, thereby preceding the development of EC senescence. The repeated addition of the NO donor S-nitroso-penicillamine significantly reduced EC senescence and delayed age-dependent inhibition of telomerase activity, whereas inhibition of endogenous NO synthesis had an adverse effect. Taken together, our results demonstrate that telomerase inactivation precedes EC aging. NO prevents age-related downregulation of telomerase activity and delays EC senescence.

A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein.

The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of ϵ‐NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N‐terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivoor in vitro. In cells, degradation of the lysine‐less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild‐type MyoD, in which all the internal Lys residues have been retained but the N‐terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell‐free system, ATP and multiple ubiquitination are essential for degradation of the lysine‐less protein. Specific chemical modifications have yielded similar results. Selective blocking of the α‐NH2 group of wild‐type protein renders it stable, while modification of the internal Lys residues with preservation of the free N‐terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N‐terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.

Pro-atherogenic factors induce telomerase inactivation in endothelial cells through an Akt-dependent mechanism

Advanced aging may contribute to impairment of angiogenesis and development of vascular diseases. Telomerase was shown to delay endothelial cell (EC) senescence. Therefore, we determined the regulation of telomerase activity in EC. Inhibition of phosphoinositol 3‐kinase (PI3K) suppressed telomerase activity, whereas inhibitors directed against ERK1/2 or protein kinase C had no effect. Dominant negative Akt significantly reduced telomerase activity. Moreover, pro‐atherogenic stimuli such as oxidized low density lipoprotein led to an inactivation of Akt and diminished telomerase activity. Thus, the PI3K/Akt pathway plays an important role in the regulation of telomerase activity. Pro‐atherosclerotic factors impair telomerase activity and thereby may promote EC aging.

Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

Degradation of MyoD by the ubiquitin pathway: regulation by specific DNA-binding and identification of a novel site for ubiquitination

MyoD is a tissue-specific transcriptional activator involvd in skeletal muscle differentiation. It is induced during transition from proliferating, non-differentiated myoblasts to the resting and well differentiated myotubes. Like many other transcriptional regulators, it is short-lived, however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved have remained obscure. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In cells, degradation is inhibited by lactacystin, a specific inhibitor of the 20S proteasome. Inhibition is accompanied by accumulation of MyoD-ubiquitin conjugates. In a cell free system, the proteolytic process requires both ATP and ubiquitin and is preceded by formation of MyoD-ubiquitin adducts. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds. Analysis of the ubiquitination site has revealed that the N-terminal residue of MyoD is sufficient and essential to promote conjugation and subsequent degradation of the protein: conjugation to internal Lys residues is not necessary. Substitution of all Lys residues did not affect significantly its degradation either in intact cells or in a reconstituted cell free system. Degradation was inhibited by specific proteasome inhibitors and was accompanied by accumulation of ubiquitinated species of the protein. We concluded that the first ubiquitin moiety is attached via its C-terminal Gly to the N-terminal residue of MyoD, and the polyubiquitin chain is then synthesized on Lys48 of this moiety.

The N-terminal domain of MyoD is necessary and sufficient for its nuclear localization-dependent degradation by the ubiquitin system

A growing number of proteins, including the myogenic transcription factor MyoD, are targeted for proteasomal degradation after N-terminal ubiquitination (NTU) where the first ubiquitin moiety is conjugated to the N-terminal residue rather than to an internal lysine. NTU might be essential in targeting both lysine-containing and naturally occurring lysine-less proteins such as p16INK4a and p14ARF; however, the mechanisms that underlie this process are largely unknown. Specifically, the recognition motif(s) in the target substrates and the ubiquitin ligase(s) that catalyze NTU are still obscure. Here we show that the N-terminal domain of MyoD is critical for its degradation and that its destabilizing effect depends on nuclear localization of the protein. Deletion of the first 15 aa of MyoD blocked completely its lysine-independent degradation. Importantly, transfer of the first 30 N-terminal residues of MyoD to GFP destabilized this otherwise stable protein, and, here too, targeting for degradation depended on localization of the protein to the nucleus. Deletion of the N-terminal domain of lysine-less MyoD did not abolish completely ubiquitination of the protein, suggesting that this domain may be required for targeting the protein also in a postubiquitination step. Interestingly, NTU is evolutionarily conserved: in the yeast Saccharomyces cerevisiae lysine-less (LL) MyoD is degraded in a ubiquitin-, N-terminal domain-, and nuclear localization-dependent manner. Taken together, our data suggest that a short N-terminal segment of MyoD is necessary and sufficient to render MyoD susceptible for ubiquitin- and nuclear-dependent degradation.