Induction of protein degradation in skeletal muscle by a phorbol ester involves upregulation of the ubiquitin-proteasome proteolytic pathway

Although muscle atrophy is common to a number of disease states there is incomplete knowledge of the cellular mechanisms involved. In this study murine myotubes were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to evaluate the role of protein kinase C (PKC) as an upstream intermediate in protein degradation. TPA showed a parabolic dose-response curve for the induction of total protein degradation, with an optimal effect at a concentration of 25 nM, and an optimal incubation time of 3 h. Protein degradation was attenuated by co-incubation with the proteasome inhibitor lactacystin (5 μM), suggesting that it was mediated through the ubiquitin-proteasome proteolytic pathway. TPA induced an increased expression and activity of the ubiquitin-proteasome pathway, as evidenced by an increased functional activity, and increased expression of the 20S proteasome α-subunits, the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme E214k, also with a maximal effect at a concentration of 25 nM and with a 3 h incubation time. There was also a reciprocal decrease in the cellular content of the myofibrillar protein myosin. TPA induced activation of PKC maximally at a concentration of 25 nM and this effect was attenuated by the PKC inhibitor calphostin C (300 nM), as was also total protein degradation. These results suggest that stimulation of PKC in muscle cells initiates protein degradation through the ubiquitin-proteasome pathway. TPA also induced degradation of the inhibitory protein, I-κBα, and increased nuclear accumulation of nuclear factor-κB (NF-κB) at the same time and concentrations as those inducing proteasome expression. In addition inhibition of NF-κB activation by resveratrol (30 μM) attenuated protein degradation induced by TPA. These results suggest that the induction of proteasome expression by TPA may involve the transcription factor NF-κB. © 2005 Elsevier Inc. All rights reserved.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.