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Autophagy plays a dual role in coronavirus infection, facilitating the elimination of either proviral components (virophagy) or antiviral factors such as mitochondria (mitophagy), leading to complex mechanisms of immune evasion. Understanding the mechanisms that govern the switch between the autophagic degradation of deleterious or beneficial substrates in coronavirus infection is crucial for developing precise drug targets to treat virus-induced diseases. However, this switch remains largely unknown. Using a dual split-fluorescence assay, we identify PDPK1 as a negative regulator of innate immunity, directing the transition from virophagy to mitophagy through the phosphorylation of SQSTM1 at T138. Remarkably, a PDPK1-targeting peptide inhibits the replication of various RNA viruses by restoring innate immunity through enhanced virophagy and suppressed mitophagy, thereby protecting female mice from lethal infections. These findings underscore the detrimental role of PDPK1 in innate immunity by orchestrating the shift from virophagy to mitophagy, positioning PDPK1 as a promising pharmacological target for effectively combating a broad spectrum of virus infections.Autophagy plays a dual role in coronavirus infection. Here, the authors identify PDPK1 as a key regulator that switches between virophagy and mitophagy and demonstrate that a PDPK1-targeting peptide restored immunity and protected mice from lethal viral infections. [ABSTRACT FROM AUTHOR]

Background: Niemann-Pick disease type C (NPC) is an inherited disorder characterized by a functional deficiency of cholesterol transport proteins. However, the molecular mechanisms and pathophysiology of the disease remain unknown. Methods: In this study, we identified several metabolite characteristics of NPC that may fluctuate in a cellular model of the disease, using both global and targeted metabolomic analyses by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Three cell lines, HepG2 cells (wild-type[WT]) and two NPC model HepG2 cell lines in which NPC1 was genetically ablated (knockout [KO]1 and KO2), were used for metabolomic analysis. Data were subjected to enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Results: The enrichment analysis of global metabolomics revealed that 8 pathways in KO1 and 16 pathways in KO2 cells were notably altered. In targeted metabolomics for 15 metabolites, 4 metabolites in KO1 and 10 metabolites in KO2 exhibited statistically significant quantitative changes in KO1 or KO2 relative to WT. Most of the altered metabolites were related to creatinine synthesis and cysteine metabolism pathways. Conclusions: In the future, our objective will be to elucidate the relationship between these metabolic alterations and pathophysiology. [ABSTRACT FROM AUTHOR]

Lysosomal storage diseases (LSDs) are caused by the deficient activity of a lysosomal hydrolase or the lack of a functional membrane protein, transporter, activator, or other protein. Lysosomal enzymes break down macromolecular compounds, which contribute to metabolic homeostasis. Stored, undegraded materials have multiple effects on cells that lead to the activation of autophagy and apoptosis, including the toxic effects of lyso-lipids, the disruption of intracellular Ca2+ ion homeostasis, the secondary storage of macromolecular compounds, the activation of signal transduction, apoptosis, inflammatory processes, deficiencies of intermediate compounds, and many other pathways. Clinical observations have shown that carriers of potentially pathogenic variants in LSD-associated genes and patients affected with some LSDs are at a higher risk of cancer, although the results of studies on the frequency of oncological diseases in LSD patients are controversial. Cancer is found in individuals affected with Gaucher disease, Fabry disease, Niemann-Pick type A and B diseases, alfa-mannosidosis, and sialidosis. Increased cancer prevalence has also been reported in carriers of a potentially pathogenic variant of an LSD gene, namely CLN3, SGSH, GUSB, NEU1, and, to a lesser extent, in other genes. In this review, LSDs in which oncological events can be observed are described. [ABSTRACT FROM AUTHOR]

Niemann-Pick disease type C1 (NP-C1) is a rare and devastating recessive inherited lysosomal lipid and cholesterol storage disorder caused by mutations in the NPC1 or NPC2 gene. These two proteins bind to cholesterol and cooperate in endosomal cholesterol transport. Characteristic clinical manifestations of NPC1 include hepatosplenomegaly, progressive neurodegeneration, and ataxia. While the rarity of NP-C1 presents a significant obstacle to progress, researchers have developed numerous potential therapeutic approaches over the past two decades to address this condition. Various methods have been proposed and continuously improved to slow the progression of NP-C1, although they are currently at an animal or clinical experimental stage. This overview of NPC1 therapy will delve into different theoretical treatment strategies, such as small molecule therapies, cell-based approaches, and gene therapy, highlighting the complex therapeutic challenges associated with this disorder. [ABSTRACT FROM AUTHOR]

Lysosomes are acidic organelles involved in crucial intracellular functions, including the degradation of organelles and protein, membrane repair, phagocytosis, endocytosis, and nutrient sensing. Given these key roles of lysosomes, maintaining their homeostasis is essential for cell viability. Thus, to preserve lysosome integrity and functionality, cells have developed a complex intracellular system, called lysosome quality control (LQC). Several stressors may affect the integrity of lysosomes, causing Lysosomal membrane permeabilization (LMP), in which membrane rupture results in the leakage of luminal hydrolase enzymes into the cytosol. After sensing the damage, LQC either activates lysosome repair, or induces the degradation of the ruptured lysosomes through autophagy. In addition, LQC stimulates the de novo biogenesis of functional lysosomes and lysosome exocytosis. Alterations in LQC give rise to deleterious consequences for cellular homeostasis. Specifically, the persistence of impaired lysosomes or the malfunctioning of lysosomal processes leads to cellular toxicity and death, thereby contributing to the pathogenesis of different disorders, including neurodegenerative diseases (NDs). Recently, several pieces of evidence have underlined the importance of the role of lysosomes in NDs. In this review, we describe the elements of the LQC system, how they cooperate to maintain lysosome homeostasis, and their implication in the pathogenesis of different NDs. [ABSTRACT FROM AUTHOR]

Acid exonuclease activity of rat liver has been determined with the help of DNA core as substrate, The intracellular distribution of the enzyme has been investigated by differential centrifugation of homogenates and compared with those of cytochrome oxidase, acid phosphatase and glucose-6-phosphatase used as reference enzymes for mitochondria, lysosomes and endoplasmic reticulum respectively. Acid exonuclease is principally associated with mitochondrial fractions and exhibits a distribution pattern similar to that of acid phosphatase. After isopyenic centrifugation in a sucrose gradient of a total mitoehondrial fraction, acid exonuclease like acid phosphatase shows a relatively flattened distribution curve with a median equilibrium density of about 1.2. The injection to the rat of Triton WR -1339, which lowers the equilibrium density of lysosomal enzymes has a similar effect on acid exonuclease. Acid exonuclease exhibits the phenomenom of structurelinked latency. it is concluded that acid exonuclease is associated with the lysosomes in rat liver. Separation between acid DNase and acid exonuelease associated with lysosomes can be performed by chromatography on hydroxyapatite of an extract of purified granules. The complementary action of the two enzymes in lysosomal digestion of DNA is illustrated, [ABSTRACT FROM AUTHOR]

It has been suggested that intracellular Hyal-1 (hyaluronidase-1), which is considered a lysosomal enzyme, originates via endocytosis of the serum enzyme. To test this proposal we have investigated the uptake and intracellular distribution of rhHyal-1 (recombinant human Hyal-1) by mouse liver, making use of centrifugation methods. Experiments were performed on wild-type mice injected with 125I-labelled rhHyal-1 and on Hyal-1-/- mice injected with the unlabelled enzyme, which were killed at various times after injection. Activity of the unlabelled enzyme was determined by zymography. Intracellular distribution of Hyal-1 was investigated by differential and isopycnic centrifugation. The results of the study indicated that rhHyal-1 is endocytosed by the liver, mainly by sinusoidal cells, and follows the intracellular pathway described for many endocytosed proteins that are eventually located in lysosomes. However, Hyal-1 endocytosis has some particular features. First, endocytosed rhHyal-1 is quickly degraded. Secondly, its distribution, as analysed by differential centrifugation, differs from the distribution of beta-galactosidase, taken as the reference lysosomal enzyme. Further analysis by isopycnic centrifugation in a sucrose gradient shows endocytosed rhHyal-1 behaves like beta-galactosidase shortly after injection. However the Hyal-1 distribution is markedly less affected than beta-galactosidase, following a prior injection of Triton WR-1339, which is a specific density perturbant of lysosomes. The behaviour in centrifugation of endogenous liver Hyal-1, identified by hyaluronan zymography, exhibits some similarity with the behaviour of the endocytosed enzyme, suggesting that it could originate from endocytosis of the serum enzyme. Overall, these results can be explained by supposing that active endocytosed Hyal-1 is mainly present in early lysosomes. Although its degradation half-time is short, Hyal-1 could exert its activity due to a constant supply of active molecules from the blood.

A number of studies, mostly performed ex vivo, suggest that lysosomes are involved in apoptosis as a result of a release of their cathepsins into the cytosol. These enzymes could then contribute to the permeabilization of the outer mitochondrial membrane; they could also activate effector caspases. The present study aims at testing whether the membrane of liver lysosomes is disrupted during Fas-mediated cell death of hepatocytes in vivo, a process implicated in several liver pathologies. Apoptosis was induced by injecting mice with aFas (anti-Fas antibody). The state of lysosomes was assessed by determining the proportion of lysosomal enzymes (beta-galactosidase, beta-glucuronidase, cathepsin C and cathepsin B) present in homogenate supernatants, devoid of intact lysosomes, and by analysing the behaviour in differential and isopycnic centrifugation of beta-galactosidase. Apoptosis was monitored by measuring caspase 3 activity (DEVDase) and the release of sulfite cytochrome c reductase, an enzyme located in the mitochondrial intermembrane space. Results show that an injection of 10 microg of aFas causes a rapid and large increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. This modifies neither the proportion of unsedimentable lysosomal enzyme in the homogenates nor the behaviour of lysosomes in centrifugation. Experiments performed with a lower dose of aFas (5 microg) indicate that unsedimentable lysosomal hydrolase activity increases in the homogenate after injection but with a marked delay with respect to the increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. Comparative experiments ex vivo performed with Jurkat cells show an increase in unsedimentable lysosomal hydrolases, but much later than caspase 3 activation, and a release of dipeptidyl peptidase III and DEVDase into culture medium. It is proposed that the weakening of lysosomes observed after aFas treatment in vivo and ex vivo results from a necrotic process that takes place late after initiation of apoptosis.

Autophagy is a biological phenomenon whereby components of cells can self-degrade using autophagosomes. During this process, cells can clear dysfunctional organelles or unwanted elements. Autophagy can recycle unnecessary biomolecules into new components or sometimes, even destroy the cells themselves. This cellular process was first observed in 1962 by Keith R. Porter et al. Since then, autophagy has been studied for over 60 years, and much has been learned on the topic. Nevertheless, the process is still not fully understood. It has been proven, for example, that autophagy can be a positive force for maintaining good health by removing older or damaged cells. By contrast, autophagy is also involved in the onset and progression of various conditions caused by pathogenic infections. These diseases generally involve several important organs in the human body, including the liver, kidney, heart, and central nervous system. The regulation of the defects of autophagy defects may potentially be used to treat some diseases. This review comprehensively discusses recent research frontiers and topics of interest regarding autophagy-related diseases. [ABSTRACT FROM AUTHOR]

Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. Lysosomal dysfunction plays a central role in the pathobiology of Parkinson's disease (PD). Gain-of-function mutations in Leucine-rich repeat kinase 2 (LRRK2) cause familial PD and genetic variations in its locus increase the risk of developing the sporadic form of the disease. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by LRRK2), wherein membrane-damaged lysosomes generate tubules sorted into mobile vesicles. Subsequently, these vesicles interact with healthy lysosomes. LYTL is orchestrated by LRRK2 kinase activity, via the recruitment and phosphorylation of a subset of RAB GTPases. Here, we summarize the current understanding of LYTL and its regulation, as well as the unknown aspects of this process. [ABSTRACT FROM AUTHOR]

Hyaluronan (HA) is a large polysaccharide that is broadly distributed and highly abundant in the soft connective tissues and embryos of vertebrates. The constitutive turnover of HA is very high, estimated at 5 g per day in an average (70 kg) adult human, but HA turnover must also be tightly regulated in some processes. Six genes encoding homologues to bee venom hyaluronidase (HYAL1, HYAL2, HYAL3, HYAL4, HYAL6P/HYALP1, SPAM1/PH20), as well as genes encoding two unrelated G8-domain-containing proteins demonstrated to be involved in HA degradation (CEMIP/KIAA1199, CEMIP2/TMEM2), have been identified in humans. Of these, only deficiencies in HYAL1, HYAL2, HYAL3 and CEMIP have been identified as the cause or putative cause of human genetic disorders. The phenotypes of these disorders have been vital in determining the biological roles of these enzymes but there is much that is still not understood. Deficiencies in these HA-degrading proteins have been created in mice and/or other model organisms where phenotypes could be analyzed and probed to expand our understanding of HA degradation and function. This review will describe what has been found in human and animal models of hyaluronidase deficiency and discuss how this has advanced our understanding of HA's role in health and disease. [ABSTRACT FROM AUTHOR]

Glucagon's ability to promote hepatic glucose production has been known for over a century, with initial observations touting this hormone as a diabetogenic agent. However, glucagon receptor agonism [when balanced with an incretin, including glucagon-like peptide 1 (GLP-1) to dampen glucose excursions] is now being developed as a promising therapeutic target in the treatment of metabolic diseases, like metabolic dysfunction-associated steatotic disease/metabolic dysfunction-associated steatohepatitis (MASLD/MASH), and may also have benefit for obesity and chronic kidney disease. Conventionally regarded as the opposing tag-team partner of the anabolic mediator insulin, glucagon is gradually emerging as more than just a "catabolic hormone." Glucagon action on glucose homeostasis within the liver has been well characterized. However, growing evidence, in part thanks to new and sensitive "omics" technologies, has implicated glucagon as more than just a "glucose liberator." Elucidation of glucagon's capacity to increase fatty acid oxidation while attenuating endogenous lipid synthesis speaks to the dichotomous nature of the hormone. Furthermore, glucagon action is not limited to just glucose homeostasis and lipid metabolism, as traditionally reported. Glucagon plays key regulatory roles in hepatic amino acid and ketone body metabolism, as well as mitochondrial turnover and function, indicating broader glucagon signaling consequences for metabolic homeostasis mediated by the liver. Here we examine the broadening role of glucagon signaling within the hepatocyte and question the current dogma, to appreciate glucagon as more than just that "catabolic hormone." [ABSTRACT FROM AUTHOR]

Around 1 in 7 men will be diagnosed with prostate cancer during their lifetime. Many strides have been made in the understanding and treatment of this malignancy over the years, however, despite this; treatment resistance and disease progression remain major clinical concerns. Recent evidence indicate that autophagy can affect cancer formation, progression, and therapeutic resistance. Autophagy is an evolutionarily conserved process that can remove unnecessary or dysfunctional components of the cell as a response to metabolic or environmental stress. Due to the emerging importance of autophagy in cancer, targeting autophagy should be considered as a potential option in disease management. In this review, along with exploring the advances made on understanding the role of autophagy in prostate carcinogenesis and therapeutics, we will critically consider the conflicting evidence observed in the literature and suggest how to obtain stronger experimental evidence, as the application of current findings in clinical practice is presently not viable. [ABSTRACT FROM AUTHOR]

Past research has identified that cancer cells sustain several cancer hallmarks by impairing function of the endolysosomal system (ES). Thus, maintaining the functional integrity of endolysosomes is crucial, which heavily relies on two key protein families: soluble hydrolases and endolysosomal membrane proteins. Particularly members of the TPC (two-pore channel) and TRPML (transient receptor potential mucolipins) families have emerged as essential regulators of ES function as a potential target in cancer therapy. Targeting TPCs and TRPMLs has demonstrated significant impact on multiple cancer hallmarks, including proliferation, growth, migration, and angiogenesis both in vitro and in vivo. Notably, endosomes and lysosomes also actively participate in various immune regulatory mechanisms, such as phagocytosis, antigen presentation, and the release of proinflammatory mediators. Yet, knowledge about the role of TPCs and TRPMLs in immunity is scarce. This prompts a discussion regarding the potential role of endolysosomal ion channels in aiding cancers to evade immune surveillance and destruction. Specifically, understanding the interplay between endolysosomal ion channels and cancer immunity becomes crucial. Our review aims to comprehensively explore the current knowledge surrounding the roles of TPCs and TRPMLs in immunity, whilst emphasizing the critical need to elucidate their specific contributions to cancer immunity by pointing out current research gaps that should be addressed. [ABSTRACT FROM AUTHOR]

The pathogenesis of acute and chronic pancreatitis has recently evolved as new findings demonstrate a complex mechanism operating through various pathways. In this review, the current evidence indicating that several mechanisms act in concert to induce and perpetuate pancreatitis were presented. As autophagy is now considered a fundamental mechanism in the pathophysiology of both acute and chronic pancreatitis, the fundamentals of the autophagy pathway were discussed to allow for a better understanding of the pathophysiological mechanisms of pancreatitis. The various aspects of pathogenesis, including trypsinogen activation, ER stress and mitochondrial dysfunction, the implications of inflammation, and macrophage involvement in innate immunity, as well as the significance of pancreatic stellate cells in the development of fibrosis, were also analyzed. Recent findings on exosomes and the miRNA regulatory role were also presented. Finally, the role of autophagy in the protection and aggravation of pancreatitis and possible therapeutic implications were reviewed. [ABSTRACT FROM AUTHOR]

Background: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection., Methods: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA., Results: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time., Conclusions: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.

Intravenous injection of gelonin and deglycosylated gelonin led to rapid clearance from the blood. Both molecules distributed similarly in liver and kidney suggesting that they followed the same pathway. Deglycosylation reduced the uptake by a third in liver, but did not affect uptake by kidney. Studies with Triton WR1339 showed a classical lysosomal pathway for both molecules. The deglycosylated molecule was degraded to a greater extent than native gelonin as seen by the presence of acid soluble radioactivity. Cell separation showed that while endothelial cells mainly took up native gelonin, Kupffer cells took up the deglycosylated molecule.

We found a dipeptidyl aminopeptidase activity in the parasitic protozoan Giardia lamblia with properties similar to the lysosomal cathepsin C of rat-liver lysosomes. Subcellular fractionation of this parasite indicated that the cathepsin C activity is located in organelles not distinguishable from the ones containing acid phosphatase, a known marker enzyme of Giardia lysosome-like peripheral vesicles. Contrary to the rat lysosomal enzyme, Giardia cathepsin C behaved like a membrane protein. Moreover, the enzyme was not solubilized by Triton X-100 or Triton X-100/SDS at 0 degrees C but could be substantially solubilized by octylglucoside, Triton X-100 at 37 degrees C or by a pretreatment with the cholesterol complexing agent beta-cyclodextrin before the Triton/SDS treatment carried out at 0 degrees C. These observations suggest that binding/anchorage of this enzyme to membranes occurs in cholesterol-rich microdomains.

Background: An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (hydrodynamics-based transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA., Methods: We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes)., Results: (35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face., Conclusions: The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA., (Copyright 2002 John Wiley & Sons, Ltd.)

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.

The cholesterol complexing agent methyl-cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl-L-phenylalanine 2-naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

Branched polyamines are extensively used as nonviral vectors for plasmid DNA in transfection experiments. Moreover, recently it has been shown that these compounds are able to eliminate prions from infected cells in cultures. It has been proposed that in both cases endosomes or lysosomes are the site of action. This raises the question of how these molecules are taken up by the cells and what is their intracellular fate. In the work presented here, the question has been addressed by investigating the uptake and the intracellular distribution of branched polyethyleneimine (25 kD) by centrifugation methods. The polyamine was labelled with (125)I-tyramine cellobiose and injected to the rat. The radioactive polymer is taken up after injection into the liver, kidney, spleen, and lungs and remains in these organs for many days. In the liver, it is found mainly in the hepatocytes. Intracellular distribution of radioactivity present in that organ was investigated by differential and isopycnic centrifugations. Early after injection, radioactivity exhibits a distribution pattern similar to that of alkaline phosphodiesterase, a plasma membrane marker. Later, the distribution pattern becomes similar to that of cathepsin C, a lysosomal enzyme. Radioactivity and hydrolase distributions in a sucrose gradient are similarly modified by a pretreatment of the rat with Triton-WR1339, a specific density perturbant of lysosomes. These results indicate that polyethyleneimine is endocytosed and reaches lysosomes. For many days it persists in these organelles probably due to its resistance to lysosomal hydrolases., (Copyright 2000 Academic Press.)

Plasmid DNA, naked or bound to a non-viral vector, is taken up by endocytosis. As a result, it has to travel through the intracellular endocytic pathway involving endosomes and lysosomes. However, some DNA molecules must escape these organelles to reach the nucleus where transcription takes place. In this paper, we consider different factors that could affect the trafficking of plasmid DNA and influence transfection efficiency.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.

Efficiency of transfection is probably dependent on the rate of intracellular degradation of plasmid DNA. When a non-viral vector is used, it is not known to what extent the plasmid DNA catabolism is subordinated to the catabolism of the vector. In the work reported here, the problem was approached by following the intracellular fate in rat liver, of plasmid [35S]DNA complexed with a cationic peptide poly-L-lysine that can be hydrolyzed by cellular peptidases or with its stereoisomer, poly-D-lysine, that cannot be split by these enzymes. Complexes of DNA with poly-L-lysine and poly-D-lysine are taken up to the same extent by the liver, mainly by Kupffer cells, but the intracellular degradation of nucleic acid molecules is markedly quicker when poly-L-lysine is injected. The association of DNA with the polycations inhibits DNA hydrolysis in vitro by purified lysosomes but similarly for poly-L-lysine and poly-D-lysine. The intracellular journey followed by [35S]DNA complexed with poly-L- or poly-D-lysine was investigated using differential and isopycnic centrifugation. Results indicate that [35S]DNA is transferred more slowly to lysosomes, the main site of intracellular degradation of endocytosed macromolecules, when it is given as a complex with poly-D-lysine than with poly-L-lysine. They suggest that the digestion of the vector in a prelysosomal compartment is required to allow endocytosed plasmid DNA to rapidly reach lysosomes. Such a phenomenon could explain why injected plasmid DNA is more stable in vivo when it is associated with poly-D-lysine.

We have studied the localization of mutant cystic fibrosis transmembrane regulator delta F508CFTR in pancreatic adenocarcinoma cells (CFPAC), which naturally express the mutant protein. Our goal was to investigate whether delta F508CFTR is strictly retained in the endoplasmic reticulum (ER) or alternatively whether it can be transported beyond the ER and reach the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). This compartment, defined by the presence of the 53-kDa protein ERGIC-53, was identified by subcellular fractionation and by immunofluorescence. Part of the delta F508CFTR population and ERGIC-53 showed similar distributions in membrane fractions analyzed on Nycodenz density gradients. Both proteins were present in density fractions distinct from the ones containing the ER marker proteins calnexin and Sec61. Immunofluorescence microscopy of CFPAC cells revealed some colocalization of delta F508CFTR with ERGIC-53. Following incubation of CFPAC cells at 15 degrees C, a condition known to block ER to Golgi transport, both ERGIC-53 and delta F508CFTR subcellular localizations were altered. By contrast, this temperature shift had no effect on the localization of the ER marker Sec61. Our observations indicate that the abnormal protein delta F508CFTR can leak out of the ER and reach the ERGIC. These results support the idea that this intermediate compartment plays a role in the trafficking events leading to retention and finally degradation of the misfolded delta F508CFTR protein.

Lysosomal-membrane-associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell-surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E-selectin and between Lamps and galectins (endogenous-galactoside-binding lectins). The present study examined the expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell-surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo-2 (human colon-adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin-3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin-3 binding to Lamps-expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis.

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N-(1-(2,3-dioleoxy)propyl)-N,N,N,-trimethylammonium methyl-sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of beta-galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.

Most lysosomal hydrolases are soluble enzymes. Lamp-II (lysosome-associated membrane protein-II) is a major constituent of the lysosomal membrane. We studied the aggregation of a series of lysosomal molecules. The aggregation-sensitive lysosomal marker enzymes were optimally aggregated at intralysosomal pH. A similar pH dependence was recorded for aggregation of Lamp-II. The pH-dependent loss of solubility of isolated Lamp-II required components of the lysosome extract. Conditions of mild acid pH promoting aggregation triggered the formation of complexes with lipids of lysosomal origin. We fractionated a membrane-free lysosome extract by gel-filtration chromatography and could reconstitute assemblies in vitro from separated fractions. We found some selectivity in the lysosomal proteins binding to complex lipids, phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine being most effective. We propose that the formation at pH 5.0 of such supramolecular assemblies between lysosomal proteins and lipids occurs within the intralysosomal environment. Some possible consequences of such an intralysosomal matrix formation on organelle function are discussed.

Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

Lamp II (for lysosomal associated membrane protein II) is an integral type I glycoprotein. It consists of a very large and heavily glycosylated luminal domain, a single transmembrane segment, and a short cytoplasmic tail. We show that in highly purified lysosomes from rat liver, Lamp II, immunodetected with a monoclonal antibody on Western blots, it surprisingly distributed between a membrane bound form and a "soluble" form. The partition of the protein between the membrane and the content of lysosomes is strongly pH dependent. The soluble Lamp II population is sensitive to pH dependent aggregation as it is for many lysosomal content enzymes.

To study the transfer of phagocytosed components from phagosomes to lysosomes, we have investigated phagocytosis by rat liver of killed Staphylococcus aureus labelled with (125)I tyramine cellobiose. Lysosomes were identified by injecting the animals with Triton WR1339, a non ionic detergent that is endocytosed by the liver and accumulates in lysosomes, causing a marked decrease of their density; that allows these organelles to be well separated from other particles in a density gradient. Bacteria were quickly taken up by the liver; their uptake is followed by a slow degradation as ascertained by the increase of acid-soluble radioactivity in the homogenates with time. Triton WR1339 injection does not affect the uptake and the degradation of the particles. Differential centrifugation of homogenates shows that at any time after injection, most of the radioactivity is recovered in the mitochondrial fractions. Distributions of acid precipitable and acid soluble radioactivities amongst subcellular structures present in mitochondrial fractions were studied by isopycnic centrifugation in sucrose gradients, at increasing times after bacteria injection. Results show that: 1) acid-precipitable radioactivity is quasi-exclusively present in gradient fractions of high density, well separated from the fractions where there are recovered lysosomes; 2) with time, acid-soluble radioactivity is more and more associated with lysosomes, however, a significant proportion can be detected for many hours after injection, in gradient fractions where acid-precipitable radioactivity is located. The most plausible explanation of our observations is that phagocytosed particles are degraded in phagosomes and that the degradation products are delivered to lysosomes, probably by a vesicular process.

Previous studies have demonstrated that reactive oxygen species are involved in ischemic injury. The present work was undertaken to determine in vivo the role of xanthine oxidase in the oxygen free radical production during rat liver ischemia and to examine the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) during the same period. Our results indicate a 4-fold increase in xanthine oxidase activity between 2 and 3 hours of normothermic ischemia, in parallel with a decrease in cell viability. Moderate hypothermia delays both events. Under the same conditions, the activity of oxygen radical scavenging enzymes remains unchanged. Moreover, we have compared in vitro the susceptibility of isolated liver cells to an oxidative stress induced by O2.-, H2O2 and .OH. Our results reveal that endothelial cells are much more susceptible to reactive oxygen species than hepatocytes, probably because they lack H2O2-detoxifying enzymes. These findings suggest that xanthine oxidase might play a major role in the ischemic injury mainly at the level of the sinusoidal space where most endothelial cells are located.

We have investigated by using centrifugation methods, the uptake and the intracellular fate of 35S DNA by rat liver and the effect on these processes of N-(1-(2,3-dioleoxyloxy)propyl)-N,N,N,-trimethylammonium-methyl-sul fate(DOTAP, Boehringer, Mannheim, Germany), an artificial cationic lipid frequently used in transfection experiments. Labeled DNA molecules are quickly taken up by the liver but a progressive degradation takes place with time. Subcellular distribution of the radioactivity was established after differential and isopycnic centrifugation. Results indicate that 35S DNA enters liver cells by endocytosis and reaches lysosomes. The uptake of 35S DNA is not modified if the molecule is associated with DOTAP but marked differences are observed after internalization of the macromolecule. When DOTAP is used, radioactive products remain for a long time in low density organelles distinct from lysosomes indicating that the transfer of internalized DNA to these organelles is delayed by the cationic lipid. These results suggest that cationic lipids could favor transfection by preventing the delivery of DNA to lysosomes, allowing these molecules to be kept intact and available for transfer from endosomes to cytosol for a long time.

Both freshly-isolated rat hepatocytes and Morris hepatoma 7777 cells synthesized cathepsin D as a precursor that was either processed intracellular to smaller mature forms or secreted into the medium. The pattern of mature enzyme forms was different in the 2 cell types. In addition, the relative amount of precursor secreted was much higher for hepatoma cells. Monensin strongly enhanced the secretion and also impaired the intracellular transport-linked maturation of procathepsin D in hepatocytes, while it markedly inhibited intracellular maturation and only slightly increased secretion of the pro-enzyme in hepatoma cells. Ammonium chloride influenced the intralysosomal segregation and maturation of procathepsin D in hepatocytes but not in hepatoma cells. Our observations indicate that (i) the lysosomal segregation of cathepsin D was less efficient and its fractional secretion higher in hepatoma cells than in hepatocytes; (ii) in the 2 cell types, delivery to lysosomes and processing of procathepsin D were differently sensitive to increases in the vacuolar pH.

The present results showed that uptake of dopamine (DA) by rat isolated hepatocytes was mediated, in addition to simple diffusion, mainly by a transporter-involved process, with Km of 66.8 mumol and Vmax of 52.3 pmol.min-1/10(5) cells. The process was pH- and temperature-dependent and required an activation energy of 4.12 kcal.mol-1 (Q10 = 1.25) in the range of 2.0-12.7 C and 13.0 kcal.mol-1 (Q10 = 2.0) in the range of 12.7-39.0 C. Cysteine residue having free thiol group was unrelated to the activity of the transporter. Catecholamines, serotonin, and cocaine inhibited the DA transport, but tyramine (TA) and tryptamine, as well as benztropine and imipramine (which are potent inhibitors for hepatic TA transporter and neuronal DA transporter), had no inhibitory effect on the transport of DA in these cells. These results indicated that DA was taken up into hepatocytes by a distinct carrier. NaF and mastoparan influenced the transport activity in these cells further, suggesting that signal transducing G-proteins may be involved in the regulation of DA transporter in rat hepatocytes.

In the work reported here, we have compared the elimination from the blood, the uptake by the liver and the intracellular distribution of bovine growth hormone, free(Gh) or bound to a monoclonal antibody (GhAb). Results show that: a) the elimination from the blood is more rapid for Gh than for GhAb; b) both molecules are quickly taken up by the liver; c) probably after travelling through endosomes, Gh and GhAb get to lysosomes where they are degraded. However, Gh mostly ends in hepatocyte lysosomes while GhAb is recovered to a large extent in sinusoidal cell lysosomes; and d) binding by isolated hepatocytes is markedly less efficient for GhAb than for Gh.

Observations on the uptake of tyramine by hepatocytes indicate that the amine is taken up by simple diffusion and a transporter mediated system, with a Km of 39 microM and a Vmax of 270 pmol/min/10(5) cells. The carrier-mediated process is pH- and temperature-dependent and requires an activation energy of 12.9 kcal/mol. An overshoot uptake is achieved a few minutes after adding this amine to the cell suspension, suggesting that active transport is involved. This is supported by the finding that partial inhibition of the uptake can be induced by oligomycin, azide, cyanide and dinitrophenol. NO3-, SCN- and SO4(2-), which change the membrane potential significantly, and depress the transporter mediated uptake further, suggesting that the membrane potential is the driving force for the entry of this amine across hepatic membrane. Cysteine is essential for the normal carrier function; whereas, histidine, tryptophan, arginine and lysine do not directly deal with the activity of the carrier. Many substances, but not amino acids, H, M, and N receptor agonists, can inhibit the uptake of tyramine. It is possible that other amines can enter hepatocytes by using this transporter.

We have examined the effect of chloroquine on rat liver lysosomal enzyme distributions after isopycnic centrifugation in a sucrose gradient. Chloroquine injection causes the large majority of cathepsin C, acid phosphatase and N acetyl glucosaminidase to migrate towards lower density regions; on the other hand only about 50% of arylsulfatase and acid deoxyribonuclease are subjected to such a density shift. To specifically mark hepatocyte lysosomes and sinusoidal cell lysosomes, rats were injected with galactosylated bovine serum albumin (A) or mannosylated bovine serum albumin (B) labelled with 125I tyramine cellobiose; A is selectively endocytosed by hepatocytes, B by sinusoidal cells. The radioactivity distribution is affected by chloroquine in the same way as cathepsin C, after injection of A though it is not influenced by chloroquine after the injection of B. These results show that chloroquine does not modify the density of liver sinusoidal cell lysosomes when it decreases the density of hepatocyte lysosomes. Such a difference could result from the fact that sinusoidal cell lysosomes do not accumulate chloroquine to the same extent as hepatocyte lysosomes. Chloroquine treatment could be useful to distinguish between hepatocyte lysosomes and sinusoidal cell lysosomes.

When a light mitochondrial fraction (L fraction) of rat liver is incubated in the presence of an oxygen free radical generating system (xanthine-xanthine oxidase), the free activity of N-acetylglucosaminidase (NAGase) increases as a result of the deterioration of the lysosomal membrane. Various flavonoids are able to prevent this phenomenon, others are ineffective. Comparative activity studies suggest the importance of the presence of two OH groups in orthosubstitution in the B ring and of an OH in the 3 position. Flavan-type flavonoids behave like their related flavonoids; d-catechin also opposes lysosome disruption. Kaempferol, quercetin, 7,8-dihydroxyflavone and d-catechin inhibit lipoperoxidation occurring in an L fraction incubated with the xanthine oxidase system as ascertained by malondialdehyde (MDA) production. For kaempferol and quercetin, such an inhibition parallels the prevention of NAGase release; this is not the case for the two other compounds where inhibition of NAGase release takes place at a flavonoid concentration lower than that required to oppose MDA production. Morphological observations performed on purified lysosomes confirm the biochemical results. Some flavonoids are also able to prevent release of NAGase caused by the incubation of an L fraction in isoosmotic glucose. Only flavone and hydroxyflavones are effective. It is proposed that the protective effect of flavonoids on lysosomes subjected to oxygen free radicals does not only originate from their scavenger and antilipoperoxidant properties; a more direct action on lysosomal membrane making it more resistant to oxidative aggression has to be considered. The prevention by some flavonoids of lysosome osmotic disruption in isoosmotic glucose could be the result of an inhibition of glucose translocation through the lysosomal membrane.

The uptake of 125I-tyramine cellobiose (TC) by isolated rat hepatocytes and by total rat liver is markedly higher than that of 14C-sucrose and 125I-PVP, suggesting that TC does not enter the cells by fluid phase endocytosis. The distribution of radioactivity after differential centrifugation shows that the compound is shared out amongst sedimentable structures and unsedimentable fraction. Analysis by isopycnic centrifugation indicates that quickly after its penetration into the cells, most of sedimentable 125I-TC is associated with lysosomes. Such an intracellular localization is confirmed by the distributions observed after free flow electrophoresis and by the fact that radioactivity and cathepsin C, a lysosomal hydrolase, are simultaneously released from a mitochondrial fraction treated with glycyl-L-phenylalanine-2-naphthylamide. Pretreatment of the rats with chloroquine, an acidotropic drug that accumulates in lysosomes, prevents to some extent the entry of 125I-TC into these organelles. Experiments performed with purified lysosomes show that 14C-sucrose does not cross the lysosomal membrane when 125I-TC accumulates linearly with time in the fractions. These results are explained by supposing that the linkage of tyramine to cellobiose allow the disaccharide to diffuse through the plasma and the lysosome membranes, and that the accumulation of the molecule in these organelles results from its weak basic properties. 125I-TC could be an interesting molecule with which to study acidotropism in the whole animal and in isolated and cultured cells.

We have investigated the endocytosis by rat liver of superoxide dismutase (SOD) labelled with 125I. (125I) SOD is quickly taken up by the liver where it remains in significant amounts for at least 150 min. Adsorptive endocytosis is probably involved. Distribution of radioactivity was established after differential and isopycnic centrifugation and compared with that of cathepsin C, a lysosomal enzyme. Results show that the behavior of radioactivity is similar to that of the hydrolase. SOD activity is only marginally affected by incubation in the presence of a purified lysosome extract; moreover, when (125I) SOD is treated in the same conditions, only a few percent of radioactivity becomes acidosoluble. These observations indicate that SOD taken up by the liver accumulates in lysosomes where it can stay for a relatively long time owing to its relative resistance to lysosomal hydrolases.

By a variety of methods, such as ultracentrifugation in different media, hypoosmotic activation and hydrostatic compression, subcellular particles were characterized at different perinatal ages and compared to the adult rats. Fetal mitochondria elicited a higher density, an increased osmotic space and a greater resistance to compression. The size of these particles was larger than in the adults. Mitochondria in the 1-day-old animals were freely permeable to sucrose and their external membrane was more resistant to hypoosmotic activation. Lysosomes were shown to decrease their sucrose permeability and their resistance to hypoosmotic activation with development. Moreover, the size of the lysosomes increased with development.