Lysosomal Degradation Products Research Articles

Lysosomal degradation products induce Coxiella burnetii virulence

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-like vacuole through activation of a Dot/Icm-type IVB secretion system and subsequent translocation of effectors that remodel the host cell. Here a genome-wide small interfering RNA screen and reporter assay were used to identify host proteins required for Dot/Icm effector translocation. Significant, and independently validated, hits demonstrated the importance of multiple protein families required for endocytic trafficking of the C. burnetii-containing vacuole to the lysosome. Further analysis demonstrated that the degradative activity of the lysosome created by proteases, such as TPP1, which are transported to the lysosome by receptors, such as M6PR and LRP1, are critical for C. burnetii virulence. Indeed, the C. burnetii PmrA/B regulon, responsible for transcriptional up-regulation of genes encoding the Dot/Icm apparatus and a subset of effectors, induced expression of a virulence-associated transcriptome in response to degradative products of the lysosome. Luciferase reporter strains, and subsequent RNA-sequencing analysis, demonstrated that particular amino acids activate the C. burnetii PmrA/B two-component system. This study has further enhanced our understanding of C. burnetii pathogenesis, the host-pathogen interactions that contribute to bacterial virulence, and the different environmental triggers pathogens can sense to facilitate virulence.

Autophagy Proteins in Phagocyte Endocytosis and Exocytosis.

Autophagy was initially described as a catabolic pathway that recycles nutrients of cytoplasmic constituents after lysosomal degradation during starvation. Since the immune system monitors products of lysosomal degradation via major histocompatibility complex (MHC) class II restricted antigen presentation, autophagy was found to process intracellular antigens for display on MHC class II molecules. In recent years, however, it has become apparent that the molecular machinery of autophagy serves phagocytes in many more membrane trafficking pathways, thereby regulating immunity to infectious disease agents. In this minireview, we will summarize the recent evidence that autophagy proteins regulate phagocyte endocytosis and exocytosis for myeloid cell activation, pathogen replication, and MHC class I and II restricted antigen presentation. Selective stimulation and inhibition of the respective functional modules of the autophagy machinery might constitute valid therapeutic options in the discussed disease settings.

Novel phenotypes of Drosophila spinster locus on the head formation during embryogenesis

spinster (spin) is a late endosome/lysosome membrane protein with the amino acid sequence of a lysosomal sugar carrier and expressed in the glial cells. Spin is required for autophagy and lysosome reformation by releasing lysosomal degradation products of autolysosome into the cytosol in Drosophila larvae and adults. However, such kind of function has not been investigated in embryos yet. In this study, for the first time, we examined the effects of spin mutation on the endocytic pathway and autophagy during embryogenesis. Loss-of-function spin mutation led to the abnormal process of early endosome/recycling endosome and the accumulation of enlarged autophagosome/autolysosome. These abnormal endocytic pathway and autophagy subsequently caused the malformation of head at embryonic stages. These results show that Spin is involved in the endocytic pathway and autophagy during embryogenesis as well as larval and adult stages.

Upregulation of macrophage-specific functions by oxidized LDL: lysosomal degradation-dependent and -independent pathways

Formation of foam cells from macrophages, which are formed by the differentiation of blood-borne monocytes, is a critical early event in atherogenesis. To examine how pre-exposure of monocytes to modified proteins, such as oxLDL, influences their differentiation to macrophages, an in vitro model system using isolated PBMC maintained in culture in the presence of oxLDL was used. Pretreatment of monocytes with oxLDL caused a faster rate of expression of macrophage-specific functions and loss of monocyte-specific functions compared to unmodified LDL. The effect of oxidation of lipid component of LDL by CuSO(4) and its protein component by HOCl, on mo-mϕ differentiation was studied by monitoring the upregulation of macrophage-specific functions, particularly MMP-9. Chloroquine, a lysosomal degradation blocker, significantly reversed the effect mediated by CuSO(4) oxLDL, indicating the involvement of lysosomal degradation products, while no such effect was observed in HOCl oxLDL-treated cells, indicating the existence of a pathway independent of its lysosomal degradation products. Reversal of the effect of oxLDL by NAC and Calphostin C, an inhibitor of PKC, suggested the activation of RO-mediated signaling pathways. Use of inhibitors of signaling pathways showed that CuSO(4) oxLDL upregulated mϕ-specific MMP-9 through p38 MAPK and Akt-dependent pathways, while HOCl oxLDL utilized ERK ½ and Akt. Further analysis showed the activation of PPARγ and AP-1 in CuSO(4) oxLDL, while HOCl-oxLDL-mediated effect involved NFκB and AP-1. These results suggest that lipid oxLDL- and protein oxLDL-mediated upregulation of mo-mϕ-specific functions involve lysosomal degradation-dependent and -independent activation of intracellular signaling pathways.

Tough antigens trigger immunity

The harder a protein is to chop up, say Delamarre and colleagues on page 2049, the more vigorous an immune response it will trigger. This suggests that the most durable antigens—or those attached to degradation-resistant carrier proteins—might make the best vaccines. Figure 1 Protease-resistant proteins (HRP) induce more robust T cell proliferation than their degradation-sensitive counterparts (apo-HRP). The correlation between stability and immunogenicity may seem counterintuitive, given that the very products of lysosomal degradation—antigenic peptides or epitopes—bind to class II MHC molecules for presentation to CD4+ T cells. And many (but not all) in vitro studies show that blocking lysosomal function in vitro inhibits antigen presentation. To determine which kind of antigen elicits the strongest immune response, Delamarre et al. stuck identical T or B cell epitopes into lysosomal degradation-resistant and degradation-sensitive versions of the same protein. Mice immunized with the more durable versions of the proteins generated more robust T cell and antibody responses than those immunized with the more degradable forms. Stabilizing the degradation-prone antigens using aldehyde fixation boosted their immunogenicity. Antigen-presenting cells did not simply have a bigger appetite for the more stable form of the protein, as both forms were taken up equally well by dendritic cells (DCs). Rather, the more stable antigens were better retained in DCs as they transited from peripheral sites of antigen capture to lymph nodes, where they present their antigenic cargo to T cells. This group previously showed that DCs express a paucity of proteases compared with macrophages, allowing them to degrade internalized proteins more slowly and thus retain them longer—perhaps explaining why DCs are so good at disseminating antigens and initiating immunity. Thus, both the nature of a protein and the cell type it ends up in help determine how immune provoking that protein will be.

Autophagy in innate and adaptive immunity against intracellular pathogens

Autophagy delivers cytoplasmic constituents for lysosomal degradation. Recent studies have demonstrated that this pathway mediates resistance to pathogens and is targeted for immune evasion by viruses and bacteria. Lysosomal degradation products, including pathogenic determinants, are then surveyed by the adaptive immune system to elicit antigen-specific T cell responses. CD4(+) T helper cells have been shown to recognize nuclear and cytosolic antigens via presentation by major histocompatibility complex (MHC) class II molecules after autophagy. Furthermore, some sources of natural MHC class II ligands display characteristics of autophagy substrates, and autophagosomes fuse with late endosomes, in which MHC class II loading is thought to occur. Although MHC class II antigen processing via autophagy has so far mainly been described for professional antigen-presenting cells like B cells, macrophages, and dendritic cells, it might be even more important for cells with less endocytic potential, like epithelial cells, when these express MHC class II at sites of inflammation. Therefore, autophagy might contribute to immune surveillance of intracellular pathogens via MHC class II presentation of intracellular pathogen-derived peptides.

Influence of NH4CI on Polarized Release of Endogenous Protein Degradation Products and on Morphology in LLC-PK1 Cells

Increased renal ammoniagenesis is thought to be a causative factor for renal hypertrophy which occurs in several disorders accompanied by metabolic acidosis. We studied the influence of ammonia on the polarized release of degradation products of endogenous proteins in LLC-PK₁ cells. Release of acid-soluble radioactivity to the extracellular space decreased under the influence of NH₄Cl with a remarkable transient reduction of basolateral release. Electron microscopically NH₄Cl-treated cells showed numerous enlarged lysosomes suggesting an accumulation of incompletely degraded cytoplasmatic material in the lysosomal compartment. We conclude that split products of lysosomal degradation of endogenous proteins are preferentially transported to the extracellular space via the basolateral plasma membrane.

Thyrotropin stimulation of lysosomal tyrosine transport in rat FRTL-5 thyroid cells.

Tyrosine countertransport was used to demonstrate the hormonal stimulation of neutral amino acid transport across the lysosomal membrane of FRTL-5 cells. Cells grown with thyrotropin (1 X 10(-10) M) had 7-fold (+/- S.E.) higher tyrosine countertransport activity in their lysosomes than cells grown without thyrotropin. Thyrotropin also stimulated the uptake into tyrosine-loaded lysosomes of other neutral amino acids recognized by the tyrosine carrier, namely, phenylalanine (3-fold) and leucine (6-fold). In contrast lysosomal cystine countertransport was not affected by thyrotropin. Addition of thyrotropin to cells grown without thyrotropin showed that the stimulation of tyrosine counter-transport (a) required at least 48 h to reach the level of the thyrotropin-supplemented cells, (b) depended upon protein synthesis, since cycloheximide (20 microM) was inhibitory, and (c) depended upon RNA synthesis, since actinomycin D (1 nM) was inhibitory. Cells grown without thyrotropin but with dibutyryl cyclic AMP (1 mM) or cholera toxin (1 nM) exhibited enhanced lysosomal countertransport of tyrosine, suggesting that cyclic AMP may act as a messenger. This represents the first demonstration of hormonal responsiveness in a lysosomal transport system and may reflect the importance of salvage and reutilization of lysosomal degradation products for the thyroid epithelial cell.

In vivo exocytosis of lysosomes by the endothelium of the venous sinuses of bone marrow and liver: visualization at normal and low body temperature.

We have visualized the exocytosis of lysosomes into the peripheral circulation by the phagocytic endothelia of the venous sinuses of liver and bone marrow of rats. Perfusion fixation at normal body temperature produced images of the earliest stages of lysosomal exocytosis. After fixation at low body temperatures (7-12 degrees C), advanced stages of this process became evident, showing extrusion of lysosomes and their contents into the circulation. It is postulated that this form of exocytosis has escaped structural detection because of its rapidity and relative infrequency as compared to merocrine secretory exocytosis, and that fixation at low body temperatures arrests or slows down these exocytic events in sufficient measure for ultrastructural visualization. The possibility that this lysosomal exocytosis contributes to the presence of lysosomal enzymes detected in the peripheral blood should be considered. In addition, it is likely that lysosomal degradation products may be discharged by exocytosis into the circulation.

Nerve growth factor-mediated induction of choline acetyltransferase in PC12 cells: evaluation of the site of action of nerve growth factor and the involvement of lysosomal degradation products of nerve growth factor

In previous experiments it has been demonstrated that nerve growth factor (NGF), subsequent to its binding to specific membrane receptors, is internalized. Ultrastructurally, this internalized NGF is localized in membrane-confined compartments which ultimately fuse with lysosomes. The present experiments were designed, first, to evaluate whether a very small but functionally important portion of the internalized NGF might reach the free cytoplasm (and subsequently the nuclear chromatin) and might be responsible for the induction of choline acetyltransferase (ChAT) in PC12 cells. Second, we investigated whether a lysosomal proteolytic degradation product of NGF might act as a second messenger in the NGF-mediated ChAT induction. In one series of experiments, guinea pig erythrocyte ghosts, loaded with NGF (or NGF antibodies), fluorescein isothiocyanate-coupled bovine serum albumin, and/or horseradish peroxidase (HRP) were fused with PC12 pheochromocytoma cells. Electron microscopy showed that [125I]NGF and HRP reaction product were located throughout the cytoplasm and the nucleus but did not penetrate membrane compartments such as the endoplasmic reticulum, the Golgi complex, the perinuclear space, or mitochondria. Biochemically, NGF injected into the cytoplasm did not produce an induction of ChAT, whereas NGF acting via cell surface receptors resulted in a 2-fold increase in ChAT. Conversely, injection of NGF antibodies did not prevent the receptor-mediated ChAT induction. In a second series of experiments, the half-life of internalized NGF was increased from 40 min to 24 hr by the administration of leupeptin, a protease inhibitor which is accumulated in lysosomes. However, the NGF-mediated ChAT induction was not affected by this treatment. It is concluded that NGF itself does not act directly on cytoplasmic or nuclear target sites, nor is a proteolytic degradation product of NGF responsible for the NGF-mediated ChAT induction. Thus, NGF must act via a second messenger mechanism, the nature of which remains to be established.

Electron-microscopic investigation of uptake of low-density lipoproteins by pericardial macrophages from patients with atherosclerosis

In atherosclerosis in man and experimental atherosclerosis in animals cells of the mononuclear phagocyte system take up lipids and are converted into "foam cells." These cells are found in organs of the reticuloendothelial system and also actually in the atherosclerotic focus in the vessel wall [i, 2, i0]. Experiments ~n ui~ro on albino rat macrophages have shown that foam cells can be formed by accumulation of products of lysosomal degradation of low-density lipoproteins (LDLP) [6]. The existence of a similar mechanism in man has also been postulated.