The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H+-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-κB in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-κB that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-κB activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-κB during infection. Our results argue that NF-κB activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-κB inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-κB in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.

The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H(+)-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.

Alterations in trafficking of cathepsins B and D have been reported in human and animal tumors. In MCF10 human breast epithelial cells, altered trafficking of cathepsin B occurs during their progression from a preneoplastic to neoplastic state. We now show that this is also the case for altered trafficking of cathepsin D. Nevertheless, the two cathepsins are not necessarily trafficked to the same vesicles. Perinuclear vesicles of immortal MCF10A cells label for both cathepsins B and D, yet the peripheral vesicles found in ras-transfected MCF10AneoT cells label for cathepsin B, cathepsin D or both enzymes. Studies at the electron microscopic level confirm these findings and show in addition surface labeling for both enzymes in the transfected cells. By immunofluorescence staining, cathepsin B can be localized on the outer surface of the cells. Similar patterns of peripheral intracellular and surface staining for cathepsin B are seen in the human breast carcinoma lines MCF7 and BT20. We suggest that the altered trafficking of cathepsins B and D may be of functional significance in malignant progression of human breast epithelial cells. Translocation of vesicles containing cathepsins B and D toward the cell periphery occurs in human breast epithelial cells that are at the point of transition between the pre-neoplastic and neoplastic state and remains part of the malignant phenotype of breast carcinoma cells.

The human neutrophil granule location of precursors of matrix metalloproteinases (MMPs), MMP-8 and -9, has been established, but that of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) has not. In this study, labeling for TIMP-1, pro-MMP-8, pro-MMP-9, and established granule marker proteins reveals that TIMP-1 is mainly located in distinct oval, electron translucent organelles, a little larger than azurophil granules. A lack of labeling for the fluid phase endocytic marker, bovine serum albumin-gold, the lysosome-associated membrane protein markers, and for glycosylphosphatidylinositol-linked proteins, which are enriched in secretory vesicles, indicates the non-endosomal, non-lysosomal, and non-secretory nature of this organelle. Density gradient cofractionation with the least dense, secretory population and some pleomorphism of the organelle suggest it is a "vesicle" rather than a "granule" population. Colocalization with pro-MMP-9 or pro-MMP-8, in minor subpopulations, suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and terminal differentiation and before secretory vesicle synthesis. Pulse-chased IgG-coated latex beads and immunolabeling show that specific and azurophil granules fuse with the phagosome whereas TIMP-1 and pro-MMP-9-containing organelles do not. This suggests that these play no role in phagosomal destruction of IgG-opsonized bacteria. Separate localization and colocalization of these proteins may, however, facilitate fine regulation of extracellular proteolysis.