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.

Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation

Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2(14k), has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2(14k) were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2(14k) mRNA and protein levels in gastrocnemius muscle with increases of 6-8-fold for C2 and two-fold for E2(14k) between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25-27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18-20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2(14k) mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss. © 2005 Cancer Research.

Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss

Atrophy of skeletal muscle is common in patients with cancer and results in increased morbidity and mortality. In order to design effective therapy the mechanism by which this occurs needs to be elucidated. Most studies suggest that the ubiquitin-proteasome proteolytic pathway is most important in intracellular proteolysis, although there have been no reports on the activity of this pathway in patients with different extents of weight loss. In this report the expression of the ubiquitin-proteasome pathway in rectus abdominis muscle has been determined in cancer patients with weight loss of 0-34% using a competitive reverse transcriptase polymerase chain reaction to measure expression of mRNA for proteasome subunits C2 and C5, while protein expression has been determined by western blotting. Overall, both C2 and C5 gene expression was increased by about three-fold in skeletal muscle of cachectic cancer patients (average weight loss 14.5 ± 2.5%), compared with that in patients without weight loss, with or without cancer. The level of gene expression was dependent on the amount of weight loss, increasing maximally for both proteasome subunits in patients with weight loss of 12-19%. Further increases in weight loss reduced expression of mRNA for both proteasome subunits, although it was still elevated in comparison with patients with no weight loss. There was no evidence for an increase in expression at weight losses less than 10%. There was a good correlation between expression of proteasome 20Sα subunits, detected by western blotting, and C2 and C5 mRNA, showing that increased gene expression resulted in increased protein synthesis. Expression of the ubiquitin conjugating enzyme, E214k, with weight loss followed a similar pattern to that of proteasome subunits. These results suggest variations in the expression of key components of the ubiquitin-proteasome pathway with weight loss of cancer patients, and suggest that another mechanism of protein degradation must be operative for patients with weight loss less than 10%. © 2004 Elsevier Ltd. All rights reserved.