No abstract

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20 -80 M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including ␣-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

No abstract

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9 -11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8 -10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and if this process is impaired, the elevated levels of aged proteins usually lead to the formation of intracellular insoluble aggregates that can cause severe pathologies (1). In mammalian cells, most proteins destined for degradation are initially tagged with a polyubiquitin chain in an energy-dependent process and then digested to small peptides by the 26 S proteasome, a large proteolytic complex involved in the regulation of cell division, gene expression, and other key processes (2, 3). In eukaryotes, 30 -90% of newly synthesized proteins may be degraded by proteasomes within minutes of synthesis (3, 4). In addition to proteasomes, other extralysosomal proteolytic systems have been reported (5, 6). The proteasome cleaves proteins into peptides that are typically 2-20 amino acids in length (7). In most cases, these peptides are thought to be rapidly hydrolyzed into amino acids by aminopeptidases (8 -10). However, some intracellular peptides escape complete degradation and are imported into the endoplasmic reticulum where they associate with major histocompatibility complex class I (MHC-I) 3 molecules and traffic to the cell surface for presentation to the immune system (10 -12). Additionally, based on the fact that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions such as gene reg...

Caffeic acid and some of its derivatives such as caffeic acid phenetyl ester (CAPE) and octyl caffeate are potent antioxidants which present important anti-inflammatory actions. The present study assessed the in vitro and in vivo effects of five caffeic acid derivatives (caffeic acid methyl, ethyl, butyl, octyl and benzyl esters) and compared their actions to those of CAPE. In the model of LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages, the pre-incubation of all derivatives inhibited nitrite accumulation on the supernatant of stimulated cells, with mean IC50 (microM) values of 21.0, 12.0, 8.4, 2.4, 10.7 and 4.80 for methyl, ethyl, butyl, octyl, benzyl and CAPE, respectively. The effects of caffeic acid derivatives seem to be related to the scavenging of NO, as the compounds prevented SNAP-derived nitrite accumulation and decreased iNOS expression. In addition, butyl, octyl and CAPE derivatives significantly inhibited LPS-induced iNOS expression in RAW 264.7 macrophages. Extending the in vitro results, we showed that the pre-treatment of mice with butyl, octyl and CAPE derivatives inhibited carrageenan-induced paw edema and prevented the increase in IL-1beta levels in the mouse paw by 30, 24 and 36%, respectively. Butyl, octyl and CAPE derivatives also prevented carrageenan-induced neutrophil influx in the mouse paw by 28, 49 and 31%, respectively. Present results confirm and extend literature data, showing that caffeic acid derivatives exert in vitro and in vivo anti-inflammatory actions, being their actions mediated, at least in part by the scavenging of NO and their ability to modulate iNOS expression and probably that of other inflammatory mediators.

Mitochondria are essential organelles for eukaryotic homeostasis. Although these organelles possess their own DNA, the vast majority (>99%) of mitochondrial proteins are encoded in the nucleus. This situation makes systems that allow the communication between mitochondria and the nucleus a requirement not only to coordinate mitochondrial protein synthesis during biogenesis but also to communicate eventual mitochondrial malfunctions, triggering compensatory responses in the nucleus. Mitochondria-to-nucleus retrograde signaling has been described in various organisms, albeit with differences in effector pathways, molecules, and outcomes, as discussed in this review.

No abstract

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance.