A locust within chromosome XIII of Saccharomyces cerevisiae containing four genes upregulated by osmotic stress has been characterized. Two of the genes, but not their osmotic induction, were already described: the DNA damage-inducible gene DDR48 and the protease inhibitor gene PAI3. The two novel genes encode a cytoplasmic aldehyde dehydrogenase (ALD2) and a peptide of unknown function (SIP18). These genes form a cluster of two pairs of divergent promoters regulated by osmotic stress. The regulation of the divergent ALD2 and DDR48 genes, however, occurs by different mechanisms. ALD2 exhibits maximum induction with 0.3 M NaCl, negative regulation by protein kinase A and dependence on PBS2 and HOG1 protein kinases for osmotic induction. DDR48 requires 1 M NaCl for maximum induction and its expression in independent of PBS2 and HOG1 protein kinases and less sensitive to protein kinase A. PAI3 and SIP18 are as dependent on the above protein kinases as ALD2. Deletion analysis indicates that most of the regulation of the ALD2 promoter is mediated by a negative element counteracted by osmotic stress.
Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b 2 of the v-type H þ ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.
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