Learn more: Proteases and Proteostasis

Oxidized mitochondrial protein degradation and repair in aging and oxidative stress
Antioxid. Redox. Signal. 2010, view full abstract in PubMed

Proteins are main targets for oxidative damage that occurs during aging and in oxidative stress situations. Since the mitochondria is a major source of reactive oxygen species, mitochondrial proteins are especially exposed to oxidative modification, and elimination of oxidized proteins is crucial for maintaining the integrity of this organelle. Hence, enzymatic reversal of protein oxidation and protein degradation is critical for protein homeostasis while protein maintenance failure has been implicated in the age-related accumulation of oxidized proteins. Within the mitochondrial matrix, the ATP-stimulated mitochondrial Lon protease is believed to play an important role in the degradation of oxidized protein, and age-associated impairment of Lon-like protease activity has been suggested to contribute to oxidized protein buildup in the mitochondria. Oxidized protein repair is limited to certain oxidation products of the sulfur-containing amino acids cysteine and methionine. Oxidized protein repair systems, thioredoxin/thioredoxin reductase or glutaredoxin/glutathione/glutathione reductase that catalytically reduce disulfide bridges or sulfenic acids, and methionine sulfoxide reductase that reverses methionine sulfoxide back to methionine within proteins, are present in the mitochondrial matrix. Thus, the role of the mitochondrial Lon protease and the oxidized protein repair system methionine sulfoxide reductase is further addressed in the context of oxidative stress and aging.

Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells
Lab Invest 2010, view full abstract in PubMed

Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis.

Protease inhibitor SERPINA1 expression in epithelial ovarian cancer
Clin. Exp. Metastasis 2010, view full abstract in PubMed

Epithelial ovarian cancer is the most lethal gynecologic cancer with a 5 years survival rate of 30-40% in patients diagnosed with high-grade invasive disease (TOV). This is in stark contrast to the 95% 5 years survival rate in ovarian cancer patients diagnosed with low malignant potential (LMP) disease. The progression from localized tumor to invasive metastasis involves matrix proteolysis. Protease inhibitors are thought to play a key role by limiting this process. Using the Affymetrix HG-U133A GeneChip array, we have studied all serine protease inhibitors and found several serpin family members that are differentially expressed between LMP and TOV serous tumors. SERPINA1 was selected for further study due to its high expression in the majority of LMP tumors and its low expression in TOV tumors; observations that were also validated by quantitative-PCR (Q-PCR). To study the effects of its over expression on different tumorigenic parameters, SERPINA1 was cloned in the pcDNA3.1+ plasmid which was subsequently used to derive stable clones from two invasive ovarian cancer cell lines, TOV-112D and TOV-1946. We found no effect of SERPINA1 over expression on tumor growth in SCID mice although cell migration and invasion were affected in in vitro assays. There was also no association between patient survival and SERPINA1 immunostaining, however, SERPINA1 localization was different in LMP (nuclear) and TOV (cytoplasmic) tumors. SERPINA1 remains an interesting candidate since protein homeostasis, regulated by proteases and their inhibitors, should be studied holistically in order to assess their full impact in tumor progression.