Regulation of human cathepsin B by alternative mRNA splicing: homeostasis, fatal errors and cell death.

One of the control mechanisms of cathepsin B biosynthesis and trafficking operates through alternative splicing of pre-mRNA. An mRNA lacking exon 2 is more efficiently translated than that containing all exons, and may be responsible for elevated biosynthesis and enzyme routing to the extracellular space, with critical consequences for connective tissue integrity in pathologies such as cancer and arthritis. mRNA missing exons 2 and 3 encodes a truncated procathepsin B form that is targeted to mitochondria.

Folding competence of N-terminally truncated forms of human procathepsin B.

Besides acting as an inhibitor, the propeptide of human cathepsin B exerts an important auxiliary function as a chaperone in promoting correct protein folding. To explore the ability of N-terminally truncated forms of procathepsin B to fold into enzymatically active proteins, we produced procathepsin B variants progressively lacking N-terminal structural elements in baculovirus-infected insect cells. N-terminal truncation of the propeptide by up to 22 amino acids did not impair the production of activable procathepsin B.

Exon skipping of cathepsin B: mitochondrial targeting of a lysosomal peptidase provokes cell death.

The alternatively spliced messenger RNA of the human cysteine peptidase cathepsin B missing exons 2 and 3 encodes a truncated form of the enzyme lacking the signal peptide and part of the inhibitory propeptide. This deletion results in a new N-terminal leader sequence characteristic of proteins predestined for transport into mitochondria. We determined enzyme targeting to intracellular organelles by transfecting HeLa cells with constructs containing segments of variable length of the N terminus of truncated cathepsin B fused to green fluorescent protein.

Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA.

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase.

The alternative use of exons 2 and 3 in cathepsin B mRNA controls enzyme trafficking and triggers nuclear fragmentation in human cells.

Pathological overexpression and trafficking of the cysteine peptidase cathepsin B depend in part on the composition of its mRNA. To investigate the roles of the alternatively spliced exons 2 and 3 in the 5'-untranslated region of cathepsin B mRNA we produced constructs of cathepsin B fused to green fluorescent protein. Expression and trafficking of the fluorescent chimeric products was followed in living human immortalized chondrocytes and HeLa cells.

Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.

Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity.