Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent

Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-alpha (TNF-alpha) cytotoxic signaling in hepatocytes and contributes to cell death. Sphingosine has recently been implicated in lysosomal permeabilization and is increased in the liver by TNF-alpha. Thus the aims of this study were to examine the mechanisms involved in TNF-alpha-associated lysosomal permeabilization, especially the role of sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent protein and LysoTracker Red were both released from lysosomes after treatment of McNtcp.24 cells with TNF-alpha/actinomycin D, a finding compatible with lysosomal destabilization. In contrast, endosomes labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-alpha or sphingosine in Cat B(+/+) but not Cat B(-/-) hepatocytes, as assessed by a fluorescence-based assay. With the use of a calcein release assay in isolated lysosomes, sphingosine permeabilized liver lysosomes isolated from Cat B(+/+) but not Cat B(-/-) liver. C(6) ceramide did not permeabilize lysosomes. In conclusion, these data implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-alpha cytotoxic signaling.     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].     

Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage

Newly-synthesized soluble lysosomal enzymes are transported from the trans-Golgi network to lysosomes by a mannose 6-phosphate receptor-mediated pathway. Lysosomal storage of indigestible material has been reported to perturb the biosynthesis and the fate of lysosomal hydrolases. In this study, we have focused our attention on the last steps in the transport of newly-synthesized cathepsin D to lysosomes in sucrose-treated WI-38 fibroblasts. Pulse-chase experiments indicate that, in sucrose-treated cells, cathepsin D maturation is delayed by 2 to 4 h. By subcellular fractionation, we show that newly-synthesized cathepsin D precursors transit through organelles endowed with a high sedimentation coefficient. These organelles are recovered in the dense region of a self-forming Percoll density gradient while the bulk of hydrolytic activities is redistributed to the low density region. Only later, are the precursors delivered to organelles containing the bulk of active hydrolases. There, procathepsin D is proteolytically processed into its 31 kDa-mature form. Our results suggest that when sucrose is present, the delayed maturation of procathepsin D is related to the delivery of the polypeptides into an organelle behaving in centrifugation like lysosomes but which is poorly efficient in proteolytic processing of procathepsin D. This low proteolytic activity of this organelle could be due to its poor ability to interact with hydrolase-containing structures.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

Intracellular uptake,transport, and processing of gold nanostructures

Abstract

The emerging field of nanomedicine requires better understanding of the interface between nanotechnology and medicine. Better knowledge of the nano-bio interface will lead to better tools for diagnostic imaging and therapy. In this review, recent progress in understanding of how size, shape, and surface properties of nanoparticles (NPs) affect intracellular fate of NPs is discussed. Gold nanostructures are used as a model system in this regard since their physical and chemical properties can be easily manipulated. The NP-uptake is dependent on the physiochemical properties, and once in the cell, most of the NPs are trafficked via an endo-lysosomal path followed by a receptor-mediated endocytosis process at the cell membrane. Within the size range of 2–100 nm, Gold nanoparticles (GNPs) of diameter 50 nm demonstrate the highest uptake. Cellular uptake studies of gold nanorods (GNRs) show that there is a decrease in uptake as the aspect ratio of GNRs increases. Theoretical models support the size- and shape-dependent NP-uptake. The intracellular transport of targeted NPs is faster than untargeted NPs. The surface ligand and charge of NPs play a bigger role in their uptake, transport, and organelle distribution. Exocytosis of NPs is dependent on size and shape as well; however, the trend is different compared to endocytosis. GNPs are now being incorporated into polymer and lipid based NPs to build multifunctional devices. A multifunctional platform based on gold nanostructures, with multimodal imaging, targeting, and therapeutics; hold the possibility of promising directions in medical research.     

Intracellular uptake, transport, and processing of gold nanostructures

The emerging field of nanomedicine requires better understanding of the interface between nanotechnology and medicine. Better knowledge of the nano-bio interface will lead to better tools for diagnostic imaging and therapy. In this review, recent progress in understanding of how size, shape, and surface properties of nanoparticles (NPs) affect intracellular fate of NPs is discussed. Gold nanostructures are used as a model system in this regard since their physical and chemical properties can be easily manipulated. The NP-uptake is dependent on the physiochemical properties, and once in the cell, most of the NPs are trafficked via an endo-lysosomal path followed by a receptor-mediated endocytosis process at the cell membrane. Within the size range of 2-100 nm, Gold nanoparticles (GNPs) of diameter 50 nm demonstrate the highest uptake. Cellular uptake studies of gold nanorods (GNRs) show that there is a decrease in uptake as the aspect ratio of GNRs increases. Theoretical models support the size- and shape-dependent NP-uptake. The intracellular transport of targeted NPs is faster than untargeted NPs. The surface ligand and charge of NPs play a bigger role in their uptake, transport, and organelle distribution. Exocytosis of NPs is dependent on size and shape as well; however, the trend is different compared to endocytosis. GNPs are now being incorporated into polymer and lipid based NPs to build multifunctional devices. A multifunctional platform based on gold nanostructures, with multimodal imaging, targeting, and therapeutics; hold the possibility of promising directions in medical research.     

Towards specific functions of lysosomal cysteine peptidases: phenotypes of mice deficient for cathepsin B or cathepsin L

The lysosomal cysteine peptidases cathepsin B and cathepsin L are abundant and ubiquitously expressed members of the papain family, and both enzymes contribute to the terminal degradation of proteins in the lysosome. However, there is accumulating evidence for specific functions of lysosomal proteases in health and disease. The generation of 'knock out' mouse strains that are deficient in lysosomal proteases provides a valuable tool for evaluation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by analysis of mice that are devoid of cathepsin B or cathepsin L. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, regulation of the hair cycle, and MHC class II-mediated antigen presentation in cortical epithelial cells of the thymus. Cathepsin B plays a major role in pathological trypsinogen activation in the early course of experimental pancreatitis and contributes significantly to TNF-alpha induced hepatocyte apoptosis.     

Monoclonal antibody toward lysosomal cathepsin B cross-reacts preferentially with distinct histone classes

1. A set of monoclonal antibodies (Mab) was prepared against cathepsin B (CB) from rat preputial-gland, an organ characterized by rapidly-renewing cell populations, which is a uniquely enriched source of lysosomal enzymes, including CB. Minute amounts of CB are known to be transferred abruptly to the nuclear compartment in a variety of activated cells. 2. Since, on the basis of its stringent substrate requirements, CB was expected to function at limited protein loci in chromatin, Mab Line II-B4 was used to probe Western blots of chromatin fractions and selected proteins. 3. The Mab, which was not directed against the active site of CB, cross-reacted preferentially with histones 3 and 4 (H3 and H4) in acid-soluble fractions of chromatin from rat preputial-gland. Line II-B4 also recognized H3 and H4 selectively in calf thymus histones and among histones purified from a wide range of sources from yeast to man. HMG 1 was minimally immunoreactive among preputial gland constituents and carbonic anhydrase (CA) was also sensitive to the Mab. 4. The common determinants were not shared by any of the H1 series, nor by H2A, H2B, protein A24 or a wide range of natural and synthetic products. 5. Origin of the antigenicity was traced by chemical modifications of H3, H4 and CA to the critical contribution of arginine and hydrophobic amino acid residues in its immediate environment, indicating that Line II-B4 may be directed against an epitope comprising the specific binding-site of CB and its selective substrate(s). 6. These data suggest that certain highly conserved cellular constituents may be uniquely vulnerable to limited proteolysis in preproliferative cells responding to mitotic signals.     

Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide

Human neutrophil lysosomal cathepsin G (cat G) exerts broad-spectrum antibacterial action in vitro against Gram-negative and -positive bacteria independent of its serine protease activity. We recently determined that an internal peptide of cat G (HPQYNQR), obtained after digestion of cat G with clostripain, possessed broad-spectrum antibacterial action in vitro, displaying an ED50 of 5 x 10(-5) M. In order to evaluate the structure-antibacterial properties of this peptide, synthetic variants with single alanine substitutions at each position were prepared and tested for antibacterial action. We found that alanine substitution for His-1 or Tyr-4, or certain modifications of the His-1 side chain, produced nonbactericidal peptides. A hexapeptide lacking the COOH-terminal Arg-7 but not a pentapeptide lacking both Gln-6 and Arg-7 possessed in vitro bactericidal activity. Interestingly, the cat G bactericidal peptide displays similarity to sequences within other serine proteases, notably the proposed cytotoxic granzymes present in the cytolytic granules of human and mouse cytotoxic T lymphocytes. We now report that an internal peptide of one human granzyme (granzyme B) with the sequence of HPAYNPK also displays bactericidal action in vitro. Our results suggest that an internal antibacterial domain among human serine proteases cat G and granzyme B has been functionally conserved through evolution perhaps for the purpose of host defense against microbial pathogens and targets of cytotoxic T lymphocyte killing.     

Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

Transformation by oncogenic ras-p21 alters the processing and subcellular localization of the lysosomal protease cathepsin D.

The expression, processing, and intracellular localization of cathepsin D (CD), an endosomal-lysosomal protease involved in malignancy, were studied in rat embryo fibroblasts transformed with an active mutant of c-Ha-ras oncogene. The pattern of the processed molecular forms of CD, comprising two single-chain mature forms of 45 and 43 kDa and two double-chain mature forms of 34 + 9 kDa and 30 + 14 kDa, expressed by the parental cell line was similar to that found in normal rat liver cells. By contrast, in the ras-transfected counterpart this pattern was profoundly altered in that the 45 kDa species was much less represented and the 30 + 14 kDa species virtually absent. In both untransformed and ras-transformed cells the conversion of proCD into mature forms was not inhibited by ammonium chloride, which is known to increase the intravacuolar pH of post-Golgi compartments. Yet, this drug induced the accumulation of the 43 and 45 kDa molecular forms of mature CD in ras-transformed cells and of the 34 kDa molecule in untransformed cells. As compared to controls, in ras-transformed fibroblasts vacuolar compartments containing CD were reduced in number and mostly located toward the periphery of the cell. This contrasted with the perinuclear distribution of CD-positive granules in untransformed cells. Serum deprivation did not affect the growth, nor the intra- and extracellular accumulation of CD activity in ras-transformed cultures, while it blocked the growth and strongly stimulated the accumulation of CD in the medium in cultures of control fibroblasts. Altogether these data are indicative for a crucial role of ras GTPase in the regulation of the transport between post-Golgi organelles.