Acidic Intracellular Compartments Research Articles

Francisella tularensis elicits a soluble factor that induces ubiquitin‐dependent MHC class II endocytosis and degradation in macrophages

The intracellular bacterium Francisella tularensis avoids degradation and replicates within macrophages (MØ), ultimately killing the host cell. Resolution of infection requires generation of IFN‐γ and development of adaptive immunity through presentation of F. tularensis antigens to CD4+ and CD8+ T cells. This laboratory has reported that F. tularensis induces MØ prostaglandin E2 (PGE2) production, leading to skewed T cell responses. PGE2 has been shown to downregulate MHC class II on MØs, suggesting that F. tularensis infection may alter T cell responses via inhibition of class II expression. To test this hypothesis, the effect of soluble factors produced by F. tularensis infected MØs on class II and CD86 expression by activated MØs was determined. Exposure of MØs to infection supernatants results in reduced surface class II and CD86. The factor eliciting class II downregulation is distinct from PGE2 and heat‐sensitive in nature. Moreover, biochemical analysis revealed a ubiquitin‐dependent mechanism of class II internalization and degradation within intracellular acidic compartments, and revealed that blocking this class II ubiquitination prevents surface clearance. These results suggest a mechanism for the modulation of MØ antigen presentation and T cell recognition during F. tularensis infection. Supported by NIH grant AI‐056320.

Synergistic antiglioma action of hyperthermia and nitric oxide

To explore combined antiglioma effect of nitric oxide (NO) and hyperthermia, the rat C6 and human U251 glioma cells were exposed to NO-releasing agents sodium nitroprusside(SNP), S-nitrosoglutathione or PAPA-NONOate, followed by hyperthermia (1 h, 43 °C). While each treatment alone showed only moderate efficiency, a synergistic cytotoxicity of NO donors and hyperthermia was clearly demonstrated by crystal violet and MTT cytotoxicity assays. The flow cytometric analysis with the appropriate reporter fluorochromes confirmed that hyperthermia and SNP cooperated in inducing oxidative stress, mitochondrial depolarization, caspase activation and DNA fragmentation, leading to both necrotic and caspase-dependent apoptotic cell death. The acridine orange staining of intracellular acidic compartments revealed that SNP completely blocked hyperthermia-induced autophagy, while the inhibition of autophagy by 3-methyl adenine mimicked SNP-triggered oxidative stress, caspase activation and cell death in hyperthermia-exposed cells. Therefore, the synergistic cytotoxicity of SNP and hyperthermia could result from NO-mediated suppression of protective autophagic response in glioma cells.

In Vivo Antitumor Activity of the Folate-Conjugated pH-Sensitive Polymeric Micelle Selectively Releasing Adriamycin in the Intracellular Acidic Compartments

Cancer treatment efficacy and safety of the environmentally sensitive polymeric micelle drug carriers were significantly increased by optimizing the number of ligands on their surface. These micelles were designed to target the cancerous tumors through the interaction between folate and its receptors that overexpress on the cancer cell membrane while achieving pH-controlled drug release in the intracellular acidic compartments such as endosomes and lysosomes. In order to elucidate the effects of folate on cytotoxicity, biodistribution, anticancer activity, and pharmacological properties, folate concentration on the surface of the micelles was controlled by precise synthesis of two different amphiphilic block copolymers that self-assemble into spherical micelles, folate-poly(ethylene glycol)-poly(aspartate-hydrazone-adriamycin) with gamma-carboxylic acid activated folate and methoxy-poly(ethylene glycol)-poly(aspartate-hydrazone-adriamycin) without folate. It is of significance that, although folate conjugation induced an extremely small change in tumor accumulation of the micelles, folate-conjugated micelles showed lower in vivo toxicity and higher antitumor activity over a broad range of the dosage from 7.50 to 26.21 mg/kg, which was 5-fold broader than free drugs.

Calcium Influx and Mitochondrial Alterations at Synapses Exposed to Snake Neurotoxins or Their Phospholipid Hydrolysis Products

Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC+FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC+FA. Here, we show that LysoPC+FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC+FA lipid mixture as a novel and effective agonist of synaptic vesicle release.

Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly

BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally degrades the low density lipoprotein receptors (LDLR). However, it is unknown whether PCSK9 acts directly on the LDLR or if PCSK9 activates another protein that in turn causes degradation of the LDLR.ResultsWe have transiently transfected HepG2 cells with wild-type and mutant D374Y-PCSK9 plasmids to study the effect of the conditioned medium on the LDLR of untransfected HepG2 cells. The ability of the conditioned medium to reduce the internalization of LDL was abolished by removal of recombinant PCSK9 from the conditioned medium by affinity chromatography. Thus, PCSK9 is the only factor in the conditioned medium able to mediate degradation of the LDLR. Moreover, fractionation of the conditioned medium by gel filtration showed that the ability of the fractions to reduce the internalization of LDL, closely paralleled the amount of D374Y-PCSK9 in the fractions. Incubation of a secreted, truncated LDLR without cytoplasmic and transmembrane domains, as well as membrane fractions from HepG2 cells, with conditioned medium containing PCSK9, did not reduce the amount of LDLR as determined by western blot analysis. Thus, the LDLR is not degraded by PCSK9 on the cell surface. The LDLR of HepG2 cells incubated with conditioned medium was protected from PCSK9-mediated degradation by the addition of nocodazole or ammonium chloride, but was not protected when the conditioned medium was made hypertonic. These findings indicate that the intracellular degradation of the LDLR involves intracellular transport along microtubules, an acidic intracellular compartment and that it occurs even when endocytosis through clathrin-coated pits has been blocked.ConclusionDegradation of the LDLR by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly. Also the PCSK9-mediated degradation of the LDLR does not take place on the cell surface. Rather, the PCSK9-mediated degradation of the LDLR appears to take place intracellularly and occurs even when endocytosis through clathrin-coated pits is blocked by hypertonic medium.

Thioredoxin reductase is required for growth and regulates entry into culmination of Dictyostelium discoideum

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase and NADPH, has been well established to be critical for the redox regulation of protein function and signalling. To investigate the role of thioredoxin reductase (Trr) in Dictyostelium discoideum, we generated mutant cells that underexpress or overexpress Trr. Trr-underexpressing cells exhibited severe defects in axenic growth and development. Trr-overexpressing (TrrOE) cells formed very tiny plaques on a bacterial lawn and had a lower rate of bacterial uptake. When developed in the dark, TrrOE cells exhibited a slugger phenotype, defined by a prolonged migrating slug stage. Like other slugger mutants, they were hypersensitive to ammonia, which has been known to inhibit culmination by raising the pH of intracellular acidic compartments. Interestingly, TrrOE cells showed defective acidification of intracellular compartments and decreased activity of vacuolar H+-ATPase which functions in the acidification of intracellular compartments. Moreover, biochemical studies revealed that the thioredoxin system can directly reduce the catalytic subunit of vacuolar H+-ATPase whose activity is regulated by reversible disulphide bond formation. Taken together, these results suggest that Dictyostelium Trr may be essential for growth and play a role in regulation of phagocytosis and culmination, possibly through the modulation of vacuolar H+-ATPase activity.

Domain Requirement for the Membrane Trafficking and Targeting of Syntaxin 1A

Syntaxin plays a key role in intracellular membrane fusion in eukaryotic cells. The function of syntaxin relies on its proper trafficking to and targeting at the target membrane. The mechanisms underlying the trafficking and targeting of syntaxin to its physiological sites remain poorly understood. Here we have analyzed the trafficking of syntaxin 1A in INS-1 and CHO cells. We have identified the transmembrane domain together with several flanking positive-charged amino acids as the minimal domain required for the membrane delivery. Interestingly, we found that SNARE motif-exposed syntaxin 1A mutants were retained in endoplasmic reticulum (ER) and failed to transport to the cell surface in the absence of SNAP-25, suggesting that the exposure of the SNARE motif causes ER retention and complexation with SNAP-25 helps the ER escape. Finally, our data propose two key roles for the H(abc) domain: to protect nonspecific interaction by masking the SNARE motif and to participate in the clustering of syntaxin 1A to the fusion sites in the plasma membrane.

Vacuolar ATPase as a Drug Discovery Target

Vacuolar ATPases (V-ATPases) are present not only in the plasma membranes of specialized cells but also in ubiquitous intracellular acidic compartments, which are essential for physiological cellular function. Consequently, although V-ATPases are important etiologically in several diseases, it seems that they might not be good molecular targets. In fact, bafilomycin A1, a potent and specific inhibitor of V-ATPase, exerts severe and acute toxic reaction when administered to animals. On the other hand, disruption of subunit a3 of V-ATPase is not embryonic lethal, but knockout mice merely exhibit osteopetrosis due to loss of osteoclastic bone resorption. In addition, recent studies have demonstrated that novel V-ATPase inhibitors, which have inhibition selectivity, can be systemically administered to animals and are highly efficacious against bone loss in lytic bone disease models. Therefore, the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is their selectivity in the inhibition.

The blood–brain barrier and treatment of lysosomal storage diseases

Lysosomes are ubiquitous, acidic intracellular compartments that fulfil a crucial role in degradation of macromolecules in cells. Specific lysosomal catabolic reactions may be impaired due to inherited defects in specific hydrolases, resulting in gradual intralysosomal accumulation of corresponding endogenous substrates. Such lysosomal deficiencies may ultimately result in devastating clinical symptoms, the so-called lysosomal storage diseases. Many of the inherited lysosomal storage disorders show phenotypic variants in which neurological complications are prominent as the result of pathological storage formation in cells inside the brain, for example perivascular macrophages, microglia or neuronal cells. In this chapter new developments and present limitations regarding therapy of (neuronopathic) lysosomal storage diseases are reviewed.

Doxorubicin loaded pH-sensitive polymeric micelles for reversal of resistant MCF-7 tumor

In order to overcome multidrug resistance in solid tumors, doxorubicin (DOX) loaded pH-sensitive micelles of which surface was decorated with folate (PHSM/f) were evaluated both in vitro and in vivo experiments. PHSM/f were fabricated from a mixture of two block copolymers of poly( l-histidine) (M n: 5K)- b-PEG (M n: 2K)-folate (polyHis/PEG-folate) (75 wt.%) and poly( l-lactic acid) (M n: 3K)- b-PEG (M n: 2K)-folate (PLLA/PEG-folate) (25 wt.%). The PHSM/f showed more than 90% cytotoxicity of DOX resistant MCF-7 (MCF-7/DOX R) when cultured with PHSM/f at a concentration of 10 μg/ml DOX. The result was interpreted by a sequential event of active internalization of PHSM/f via folate-receptor mediated endocytosis and ionization of His residues which result in micelle destabilization and probably disturbance of endosomal membranes. This potential mechanism may endow the drug carriers to bypass Pgp efflux pump and sequestration of DOX in acidic intracellular compartments, yielding high cytotyoxicity. Experimental evaluation of tumor regression was carried out in a small animal model bearing s.c. MCF-7 or MCF-7/DOX R xenografts. The tumor (MCF-7/DOX R) volumes of mice treated with PHSM/f were significantly less than control groups treated with free DOX or similar micelles but without folate (PHSM). In the MCF-7/DOX R xenograft model, the accumulated DOX level of PHSM/f in solid tumors was 20 times higher than free DOX group, and 3 times higher than PHSM group. The results demonstrate that PHSM/f is a viable means for treating drug resistant tumors.

Apoptosis-Mediated Cytotoxicity of Prodigiosin-Like Red Pigment Produced by .GAMMA.-Proteobacterium and Its Multiple Bioactivities

Recently we discovered a bacterial strain (MS-02-063) that produces large amounts of red pigment (PG-L-1). Among the cell lines tested, U937 cells showed the highest susceptibility to PG-L-1 toxicity. PG-L-1 induced typical apoptotic nuclear morphological changes, and single cell gel electrophoresis revealed that PG-L-1 caused DNA fragmentation in U937 cells. In PG-L-1 treated U937 cells, the acidic compartment such as lysosomes disappeared, suggesting that PG-L-1-induced disorder of intracellular pH compartmentalization might trigger apoptotic signal. Since p38 MAP kinase inhibitor specifically prevented the PG-L-1 mediated cell death, p38 MAP kinase may be involved in the cytotoxic mechanism. In fact, immunoblot analysis of p38 MAP kinase revealed that phosphorylation of p38 MAP kinase occurred in PG-L-1-treated U937 cells. In addition to the activity to induce apoptotic cell death as reported in several PG family members, our chemiluminescence analysis suggested that PG-L-1 inhibited superoxide generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated U937 cells in a dose-dependent manner. Since PG-L-1 had no effect on the chemiluminescence response caused by xanthine oxidase/hypoxanthine system, PG-L-1 acts on the enzyme system responsible for O(2)(-) generation rather than direct scavenging toward O(2)(-). Our results suggest that PG-L-1 causes multiple biochemical effects on the target cells such as increase in pH in acidic intracellular compartment, activation of p38 MAP kinase, inhibition of O(2)(-) generation, and eventually induces apoptotic cell death.

Fluorescence imaging method for in vivo pH monitoring during liposomes uptake in rat liver using a pH-sensitive fluorescent dye

Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6-carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel.

Entry of the enzymatic components of anthrax toxin [lethal factor (LF) and edema factor] into the cytosol of mammalian cells depends on the ability of the activated protective antigen (PA63) component to form a channel (pore) in the membrane of an acidic intracellular compartment. To investigate the mechanism of translocation, we characterized N-terminally truncated forms of the PA63-binding domain of LF (LFN). Deleting 27 or 36 residues strongly inhibited acid-triggered translocation of LFN across the plasma membrane of CHO-K1 cells and ablated the protein's ability to block PA63 channels in planar lipid bilayers at a small positive voltage (+20 mV). Fusing a H6-tag to the N terminus of the truncated proteins restored both translocation and channel-blocking activities. At +20 mV, N-terminal H6 and biotin tags were accessible to Ni2+ and streptavidin, respectively, added to the trans compartment of a planar bilayer. On the basis of these findings, we propose that the N terminus of PA63-bound LF or edema factor enters the PA63-channel under the influence of acidic pH and a positive transmembrane potential and initiates translocation in an N- to C-terminal direction.

Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils

The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1+/-0.04 to 7.5+/-0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca2+]i after PAF stimulation activates a H+-ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins.

Anthrax Protective Antigen: Efficiency of Translocation Is Independent of the Number of Ligands Bound to the Prepore

Heptameric anthrax protective antigen (termed prepore), which assembles at the mammalian cell surface, competitively binds edema factor (EF) and/or lethal factor (LF). It then transports them to an acidic intracellular compartment and mediates their translocation across the membrane to the cytosol. Steric constraints limit to three the number of molecules of EF and/or LF that can bind simultaneously to prepore. To determine whether the number of ligand molecules bound per heptamer affects the efficiency of translocation, we measured the low-pH-triggered translocation of the radiolabeled protective antigen (PA(63))-binding domain of LF ((35)S-LF(N)) across the plasma membrane of CHO-K1 cells as a function of the degree of saturation of the prepore. The fraction translocated remained constant at approximately 0.4 as (35)S-LF(N) was varied from nil through saturating concentrations. The same constant value was observed when we held (35)S-LF(N) at a saturating concentration and varied the number of functional ligand sites per prepore by changing the ratio of wild-type PA to a ligand-binding mutant. Thus, prepore containing only a single ligand-binding site is capable of translocating its cargo as efficiently as one containing multiple binding sites. The results as a whole imply that heptamers with one, two, or three ligands bound translocate their ligands with the same efficiency, indicating that each ligand molecule is translocated independently from the others.

PH Dependence and Stoichiometry of Binding to the Fc Region of IgG by the Herpes Simplex Virus Fc Receptor gE-gI

Herpes simplex virus type 1 encodes two glycoproteins, gE and gI, that form a heterodimer on the surface of virions and infected cells. The gE-gI heterodimer has been implicated in cell-to-cell spread of virus and is a receptor for the Fc fragment of IgG. Previous studies localized the gE-gI-binding site on human IgG to a region near the interface between the C(H)2 and C(H)3 domains of Fc, which also serves as the binding site for bacterial and mammalian Fc receptors. Although there are two potential gE-gI-binding sites per Fc homodimer, only one gE-gI heterodimer binds per IgG in gel filtration experiments. Here we report production of recombinant human Fc molecules that contain zero, one, or two potential gE-gI-binding sites and use them in analytical ultracentrifugation experiments to show that two gE-gI heterodimers can bind to each Fc. Further characterization of the gE-gI interaction with Fc reveals a sharp pH dependence of binding, with K(D) values of approximately 340 and approximately 930 nm for the first and second binding events, respectively, at the slightly basic pH of the cell surface (pH 7.4), but undetectable binding at pH 6.0. This strongly pH-dependent interaction suggests a physiological role for gE-gI dissociation from IgG within acidic intracellular compartments, consistent with a mechanism whereby herpes simplex virus promotes intracellular degradation of anti-viral antibodies.

Analysis of post-lysosomal compartments

Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6 h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lysosomes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-depolymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.

Plasmalemmal vacuolar-type H+-ATPase in cancer biology.

Vacuolar-type H+-adenosine triphosphatase (V-ATPase) is one of the most fundamental enzymes in nature. V-ATPases are responsible for the regulation of proton concentration in the intracellular acidic compartments. It has similar structure with the mitochondrial F0F1-ATP synthase (F-ATPase). dagger The V-ATPases are composed of multiple subunits and have various physiological functions, including membrane and organelle protein sorting, neurotransmitter uptake, cellular degradative processes, and cytosolic pH regulation. The V-ATPases have been involved in multidrug resistance. Recently, plasma membrane V-ATPases have been involved in regulation of extracellular acidity, essential for cellular invasiveness and proliferation in tumor metastasis. The current knowledge regarding the structure and function of V-ATPase and its role in cancer biology is discussed.

Biosynthesis, Glycosylation, and Enzymatic Processingin Vivo of Human Tripeptidyl-peptidase I

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.

Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death.

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-alpha)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-alpha-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-alpha in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.