Lysosomal proteases have classically been assumed to function exclusively in the cooperative mediation of terminal degradation of endocytosed and endogenous proteins within acidic late endosomes or lysosomes. Recent data, however, implicate these proteases in additional diverse processes such as antigen processing, apoptosis and extracellular matrix remodeling, suggesting the proteases may be targeted to and activated in cellular sites distinct from the lysosome. In normal cells, the lysosomal cysteine protease cathepsin L is efficiently targeted to lysosomes through interaction of its phosphorylated mannose residues with mannose phosphate receptors that recognize the protein in the TGN and transport it to late endosomes. When transcriptional regulation increases expression of the protease, whether during a normal developmental stage, as occurs in Sertoli cells during sperm maturation, or as a consequence of cell transformation, the additional protease synthesized is predominately secreted rather than targeted to lysosomes, consistent with a potential role for this protease in extracellular processes.
The goal of this project is to gain understanding of the mechanism that sifts targeting of a single protease from the lysosome to the cell exterior as a consequence of increased expression. The association of cathepsin L with two cellular molecules will be characterized, and how these molecular interactions modulate intracellular targeting of the protease will be studied. The hypothesis that will be tested is that tetraspanins, which are molecular facilitators thought to comprise a web that stabilizes and facilitates protein interaction, serve as packaging chaperones or receptors for procathepsin L. In the dense cores of multivesicular endosomes, procathepsin L colocalizes with the 43-kDa tetraspanin CD63, as demonstrated by EM immunogold labeling. By yeast two-hybrid assay, procathepsin L binds CD82, which has been shown to interact with CD63. An additional hypothesis is that procathepsin L also binds to a mammalian homologue of RMR, a RING-finger containing plant receptor that recognizes a C-terminal sorting signal in the vacuolar cysteine protease aleurain to mediate targeting of this enzyme to protein storage vacuoles in Arabidopsis thaliana. Preliminary data show that antibodies to the plant receptor recognize a single protein of correct size in mouse fibroblast microsomes, and that in a pull-down assay, the ligand-binding domain of the plant receptor binds active two-chain cathepsin L.
By epitope tag addition and introduction of point mutations, two sequences have beeen identified within procathepsin L that modulate protease targeting. One mutation causes the enzyme to accumulate in the Golgi, while the other induces accumulation in perinuclear vesicles, as assayed by immunofluorescence microscopy. This suggests that two surfaces of the molecule interact with proteins which mediate targeting. We hypothesize that the tetraspanins facilitate Golgi export of procathepsin L by interacting with the N-terminal site, while the plant receptor homologue participates in later targeting events through recognition of C-terminal sequences. We further hypothesize that these molecular interactions mediate discrete steps in the biosynthetic pathway that direct cathepsin L to multivesicular endosomes, lysosomes and the secretory pathway.
In animal cells, targeting of lysosomal enzymes to intracellular compartments occurs via at least two targeting pathways. One of these, which has been very extensively characterized, involves a specific receptor that recognizes mannose-6-phosphate (Man-6-P) residues on lysosomal enzymes and binds to them to carry them to lysosomes. The other targeting process(es) are independent of Man-6-P recognition and remain largely unknown. In plants and yeast, however, the Man-6-P pathway does not even exist, so the Man-6-P independent pathway(s) is obviously important. This project holds the promise of revealing the molecular mechanism at least one of the Man-6-P independent trafficking pathways that has eluded investigators for so many years.
BROADER IMPACT OF PROPOSED PROJECT: The proposed experiments will be utilized as teaching vehicles for both undergraduate chemistry and biology majors and biochemistry graduate students. Hands-on research not only teaches students basic technology but teaches them to think critically. They must not only learn to utilize precedent and design controls but also to determine which variables could have generated the results obtained in addition to the one which formed the basis for their original hypothesis. This critical thinking only comes with time and can't be learned from a textbook. By participating in research, and being forced to think critically about data that they have generated, students learn the thinking processes critical for success in research.
