RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit.
(1/5483)
The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t(1/2) approximately 2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4Delta cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription-activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome. (+info)
Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling.
(2/5483)
The high affinity receptor for IgE, FcepsilonRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcepsilonRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcepsilonRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcepsilonRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcepsilonRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcepsilonRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcepsilonRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts. (+info)
The Mdm-2 amino terminus is required for Mdm2 binding and SUMO-1 conjugation by the E2 SUMO-1 conjugating enzyme Ubc9.
(3/5483)
Covalent attachment of SUMO-1 to Mdm2 requires the activation of a heterodimeric Aos1-Uba2 enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm2 by Ubc9, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Upon Sumo-1 conjugation, Mdm2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide ligase (E3) activity toward p53, thereby increasing its oncogenic potential. Because of the biological implication of Mdm2 sumoylation, we mapped Ubc9 binding on Mdm2. Here we demonstrate that Ubc9 can associate with Mdm2 only if amino acids 40-59 within the N terminus of Mdm2 are present. Mdm2 from which amino acids 40-59 have been deleted can no longer be sumoylated. Furthermore, addition of a peptide that corresponds to amino acids 40-59 on Mdm2 to a sumoylation reaction efficiently inhibits Mdm2 sumoylation in vitro and in vivo. In UV-treated cells Mdm2 exhibits reduced association with Ubc9, which coincides with decreased Mdm2 sumoylation. Our findings regarding the association of Ubc9 with Mdm2, and the effect of UV-irradiation on Ubc9 binding, point to an additional level in the regulation of Mdm2 sumoylation under normal growth conditions as well as in response to stress conditions. (+info)
Heat capacity changes upon burial of polar and nonpolar groups in proteins.
(4/5483)
In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, DeltaC(p), that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn. As a control, Ala was introduced at the same three positions. We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln. As a control, Asn was introduced at Gln41 as well. The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry. The effect of the amino acid substitutions on the structure was also evaluated by comparing the (1)H-(15)N HSQC spectra of the ubiquitin variants. It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding. However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding. The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants. The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding. These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign. However, the observed changes in DeltaC(p) are several times larger than those predicted, based on the changes in water accessible surface area upon substitution. (+info)
Proteasomes are involved in the constitutive degradation of growth hormone receptors.
(5/5483)
In the mouse Ba/F3-hGHR cell line, which stably expresses human growth hormone receptors (hGHRs), the hGHRs were rapidly degraded in the absence of the ligand. Human growth hormone-binding protein (hGH-BP), a soluble form of hGHR, was released from Ba/F3-hGHR cells, but the hGH-BP release was less than 1% of total hGHRs in the cells. Therefore, the hGH-BP release does not markedly contribute to hGHR degradation in Ba/F3-hGHR cells. The constitutive degradation of hGHRs was inhibited by the proteasome inhibitors MG-132 and clasto-lactacystin beta-lactone, or the vacuolar H+-ATPase inhibitor, bafilomycin A1. hGH-enhanced degradation of hGHRs was also inhibited by MG-132. Moreover, MG-132 inhibited the internalization of hGHRs as assessed by 125I-hGH binding to the cell surfaces. Ubiquitinated hGHRs were detected in the cell lysate and increased by hGH-treatment. Furthermore, MG-132 accumulated the ubiquitinated hGHRs induced by hGH. However, the ratio of ubiquitinated hGHRs to unubiquitinated hGHRs was very small, even with treatment involving both hGH and MG-132. In the hGH-untreated cells, the ubiquitinated hGHRs were weakly detected. However, the ubiquitination of hGHR was not enhanced by MG-132 as a result of immunoblotting. Thus, the ubiquitination of hGHR is unlikely to be involved, at least in the constitutive degradation. Taken together, both the proteasome pathway and endosome/lysosome pathway are involved in the constitutive degradation of hGHRs. Our results also suggest that ubiquitination of the hGHR itself is unlikely to be the trigger of the proteasome-dependent degradation. (+info)
High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter.
(6/5483)
Sustained transgene expression will be required for the successful treatment of most genetic diseases being considered for gene therapy. The initially high levels of expression attained with plasmid DNA (pDNA) vectors containing viral promoters, such as that from cytomegalovirus (CMV), decline precipitously to near-background levels within two to three weeks. Here we constructed pDNA vectors containing the human cellular UBB (encoding ubiquitin B; Ub) promoter and evaluated their expression in the mouse lung. Cationic lipid-pDNA complexes were instilled intranasally (IN) or injected intravenously (IV) into immunodeficient BALB/c mice. Chloramphenicol acetyltransferase (CAT) reporter gene expression from the UBB promoter was initially very low at day 2 post-administration, but by day 35 exceeded the level of expression attained from a CMV promoter vector by four- to ninefold. Appending a portion of the CMV enhancer 5' of the UBB promoter (CMV-Ub) increased CAT expression to nearly that of the CMV promoter and expression persisted in the lung for at least 3 months, with 50% of day 2 levels remaining at day 84. In the liver, expression from the CMV-Ub hybrid promoter was sustained for 42 days. As previous studies have shown that eliminating immunostimulatory CpG motifs in pDNA vectors reduces their toxicity, we constructed a CpG-deficient version of the CMV-Ub vector expressing alpha-galactosidase A, the enzyme deficient in Fabry disease, a lysosomal storage disorder. After IN or IV administration, levels of alpha-galactosidase A from this vector were not only undiminished but increased 500% to 1500% by day 35. Our results indicate that CpG-reduced plasmid vectors containing a CMV-Ub hybrid promoter may provide the long-term expression required for a practical gene therapeutic. (+info)
Effects of arachidonic and docosahexaenoic acids on secretion and degradation of bile salt-dependent lipase in AR4-2J cells.
(7/5483)
In this study we demonstrated that two polyunsaturated fatty acids, arachidonic acid (AA, n-6) and docosahexaenoic acid (DHA, n-3), modulate the secretion of bile salt-dependent lipase (BSDL) by pancreatic AR4-2J cells. The effects of AA and DHA were also compared with that of the monounsaturated fatty acid, oleic acid (OA). Our results showed that the chronic treatment of cells with AA or DHA, that did not affect the biosynthesis rate of BSDL, similarly decreased the amount of secreted BSDL and perturbed the intracellular partitioning of the enzyme, whereas OA had no effect. Particularly, AA and DHA induced the retention of the enzyme in microsomes and lowered its content in the cell cytosol. We have further shown that AA treatment decreased the ubiquitination of the protein, and consequently diminished its export toward the cytosol, a result that might explain the retention of BSDL in microsomes and correlated with membrane phospholipids alteration. The retained protein was further degraded by a nonproteasomal pathway that likely involves ATP-dependent endoplasmic reticulum proteases. These findings concerning the regulation of the pancreatic BSDL secretion by two polyunsaturated acids, AA and DHA, might be of physiological importance in the plasmatic and cellular cholesterol homeostasis. (+info)
Toxoplasma gondii tachyzoites inhibit proinflammatory cytokine induction in infected macrophages by preventing nuclear translocation of the transcription factor NF-kappa B.
(8/5483)
Control of microbial infection requires regulated induction of NF-kappaB-dependent proinflammatory cytokines such as IL-12 and TNF-alpha. Activation of this important transcription factor is driven by phosphorylation-dependent degradation of the inhibitory IkappaB molecule, an event which enables NF-kappaB translocation from the cytoplasm to the nucleus. In this study, we show that intracellular infection of macrophages with the protozoan parasite Toxoplasma gondii induces rapid IkappaB phosphorylation and degradation. Nevertheless, NF-kappaB failed to translocate to the nucleus, enabling the parasite to invade cells without triggering proinflammatory cytokine induction. Infected cells subsequently subjected to LPS triggering were severely crippled in IL-12 and TNF-alpha production, a result of tachyzoite-induced blockade of NF-kappaB nuclear translocation. Our results are the first to demonstrate the ability of an intracellular protozoan to actively interfere with the NF-kappaB activation pathway in macrophages, an activity that may enable parasite survival within the host. (+info)