Acidic Subcellular Compartments Research Articles

Differences in Sustained Cellular Effects of MET inhibitors Are Driven by Prolonged Target Engagement and Lysosomal Retention.

Intracellular distribution of drug compounds is dependent on physicochemical characteristics and may have a significant bearing on the extent of target occupancy and, ultimately, drug efficacy. We assessed differences in the physicochemical profiles of MET inhibitors capmatinib, crizotinib, savolitinib, and tepotinib and their effects on cell viability and MET phosphorylation under steady-state and washout conditions (to mimic an open organic system) in a human lung cancer cell line. To examine the differences of the underlying molecular mechanisms at the receptor level, we investigated the residence time at the kinase domain and the cellular target engagement. We found that the ranking of the drugs for cell viability was different under steady-state and washout conditions and that under washout conditions, tepotinib displayed the most potent inhibition of phosphorylated MET. Postwashout effects were correlated with the partitioning of the drug into acidic subcellular compartments such as lysosomes, and the tested MET inhibitors were grouped according to their ability to access lysosomes (crizotinib and tepotinib) or not (capmatinib and savolitinib). Reversible lysosomal retention may represent a valuable intracellular storage mechanism for MET inhibitors, enabling prolonged receptor occupancy in dynamic, open-physiologic systems and may act as a local drug reservoir. The use of washout conditions to simulate open systems and investigate intracellular drug distribution is a useful characterization step that deserves further investigation. SIGNIFICANCE STATEMENT: Generally, determination of potency and receptor occupancy is performed under steady-state conditions. In vivo conditions are more complex due to concentration differences between compartments and equilibrium processes. Experiments under steady state cannot explore effects such as sustained target inhibition. This study has shown that differences between MET inhibitors are observable by applying washout conditions to in vitro assays. This important finding applies to most compound classes and may inspire readers to rethink their assay designs in the future.

Mesoporous Nanofibers Mediated Targeted Anti-cancer Drug Delivery

ABSTRACTTumor tissue has different acidity compared to normal tissue. Localized drug delivery that release chemotherapeutic medications upon stimulation via pH changes is a promising strategy in cancer therapy for adjuvant therapies after surgical resection to reduce the risk of local recurrence. In this study, a mesoporous nanofibrous system with acidic pH-triggered “caps” has been for the first time developed for localized on-demand drug release to target tumor cells, without biological damage to normal cells while maintaining their structural integrity to support future tissue regeneration. Specifically, polyacrylic acid (PAA) was grafted on electrospun mesoporous silica nanofibers (MSFs) and the obtained PAA-MSFs allowed efficient drug loading at neural pH and on-demand releasing at acidic cancer subcellular compartments, based on pH-dependent electrostatic interactions associated with protonation/deprotonation of PAA. The The hybrid mesoporous nanofibers a low cytotoxicity to normal cells and a high killing efficiency to cancer cells. The system demonstrated a great potential as tumor targeting drug delivery system.

An Albumin-binding Polypeptide Both Targets Cytotoxic T Lymphocyte Vaccines to Lymph Nodes and Boosts Vaccine Presentation by Dendritic Cells.

Rationale: Albumin-binding carriers have been shown to target cytotoxic T lymphocyte (CTL) vaccines to lymph nodes (LNs) and improve the efficacy of the vaccines. However, it was not clear whether the improved efficacy is solely due to the LN targeting, which prompted this study.Methods: First, we generated a fusion protein consisting of an albumin-binding domain (ABD) and an immune-tolerant elastin-like polypeptide (iTEP). Then, we examined the binding between this fusion protein, termed ABD-iTEP, and mouse serum albumin (MSA). Next, we evaluated the accumulation of ABD-iTEP in LNs and dendritic cells (DCs) in the LNs. We also analyzed antigen presentation and in vitro T cell activation of vaccines that were delivered by ABD-iTEP and investigated possible underlying mechanisms of the presentation and activation results. Last, we measured CTL responses induced by ABD-iTEP-delivered vaccines in vivo.Results: ABD-iTEP bound with MSA strongly with an affinity of 1.41 nM. This albumin-binding carrier, ABD-iTEP, accumulated in LNs 3-fold more than iTEP, a control carrier that did not bind with albumin. ABD-iTEP also resulted in 4-fold more accumulation in DCs in the LNs than iTEP. Most importantly, ABD-iTEP drastically enhanced the antigen presentation of its vaccine payloads and the T cell activation induced by its payloads. The enhancement was dependent on the formation of the complex between MSA and ABD-iTEP. Meanwhile, the MSA/ABD-iTEP complex was found to have increased stability in acidic subcellular compartments and increased cytosolic accumulation in DCs, which might explain the enhanced vaccine presentation resulting from the complex. Finally, when ABD-iTEP was used to deliver CTL vaccines derived from both self- and non-self-antigens, it boosted the vaccine-induced responses by 2-fold in either case.Conclusion: ABD-iTEP not only targets vaccines to LNs but also promotes the presentation of the vaccines by DCs. Albumin-binding carriers have more than one mechanism to boost the efficacy of CTL vaccines.

Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line

ABSTRACT Objective: Staphylococcus aureus is a particularly difficult pathogen to eradicate from the respiratory tract. Previous studies have highlighted the intracellular capacity of S.aureus in several phagocytic and non-phagocytic cells. The aim of this study was to define S.aureus interaction within a murine alveolar macrophage cell line. Methods: Cell line MH-S was infected with Newman strain. Molecular mechanisms involved in phagocytosis were explored. To assess whether S.aureus survives intracellularly quantitative (gentamicin protection assays and bacterial plating) and qualitative analysis (immunofluorescence microscopy) were performed. Bacterial colocalization with different markers of the endocytic pathway was examined to characterize its intracellular trafficking. Results: We found that S.aureus uptake requires host actin polymerization, microtubule assembly and activation of phosphatidylinositol 3-kinase signaling. Time course experiments showed that Newman strain was able to persist within macrophages at least until 28.5 h post infection. We observed that intracellular bacteria are located inside an acidic subcellular compartment, which co-localizes with the late endosome/lysosome markers Lamp-1, Rab7 and RILP. Colocalization counts with TMR-dextran might reflect a balance between bacterial killing and intracellular survival. Conclusions: This study indicates that S.aureus persists and replicates inside murine alveolar macrophages, representing a privileged niche that can potentially offer protection from antimicrobial activity and immunological host defense mechanisms.

PH-Sensitive Pt Nanocluster Assembly Overcomes Cisplatin Resistance and Heterogeneous Stemness of Hepatocellular Carcinoma.

Response rates to conventional chemotherapeutics remain unsatisfactory for hepatocellular carcinoma (HCC) due to the high rates of chemoresistance and recurrence. Tumor-initiating cancer stem-like cells (CSLCs) are refractory to chemotherapy, and their enrichment leads to subsequent development of chemoresistance and recurrence. To overcome the chemoresistance and stemness in HCC, we synthesized a Pt nanocluster assembly (Pt-NA) composed of assembled Pt nanoclusters incorporating a pH-sensitive polymer and HCC-targeting peptide. Pt-NA is latent in peripheral blood, readily targets disseminated HCC CSLCs, and disassembles into small Pt nanoclusters in acidic subcellular compartments, eventually inducing damage to DNA. Furthermore, treatment with Pt-NA downregulates a multitude of genes that are vital for the proliferation of HCC. Importantly, CD24+ side population (SP) CSLCs that are resistant to cisplatin are sensitive to Pt-NA, demonstrating the immense potential of Pt-NA for treating chemoresistant HCC.

Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites

A number of biological functions have been ascribed to mammalian metallothioneins (MTs) including zinc and copper homeostatic regulation, redox activity and detoxification of heavy metals like cadmium and mercury. It is unclear how these small, fluxional, cysteine rich proteins manage to play each of these vital roles. Using a combination of cadmium and pH titrations of the isolated domains of human MT isoform 1a monitored by electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report the pH dependencies that control metal binding mechanisms of these domains. We report that the α-domain mechanism is driven by the cooperative formation of the Cd4MT cluster at slightly acidic pH (≤6.9) switching binding mechanisms over a physiologically relevant pH range, whereas the β-domain metalation mechanism is dominated by terminal coordination of cadmium in a non-cooperative manner above pH5.5. These results suggest that, in some acidic sub-cellular compartments, cadmium could be sequestered in the α-domain, leaving zinc or copper bound in the β-domain and available for donation to other metalloproteins. We propose that these results can be explained by the intrinsic nature of the two domains, the four-metal α-cluster being more resistant to proton attack due to its lower charge-to-metal ratio, compared with the three-metal β-domain.

Nk-lysin, an antimicrobial peptide expressed in fish erythrocytes is involved in antiviral defense

Erythrocytes or red cells are the most abundant cells in the blood of vertebrate organisms and their primary function is the transport of oxygen. Nevertheless, it is known that these cells also play a role in some immune responses. Nk-lysin is a cationic antimicrobial peptide mainly produced by natural killer cells and cytotoxic T lymphocytes and stored in cytolytic granules together with perforin and granzymes. In this work, Nk-lysin (nkl) was characterized for the first time in turbot (Scophthalmus maximus). Interestingly, immunofluorescence analysis of head kidney and blood cell populations by flow cytometry revealed the presence of the Nkl peptide in non-lymphoid lineages. Confocal images showed that nkl was highly expressed in lymphocytes but also in erythrocytes although, in this case, the presence of Nkl was mainly confined to spherical vacuolar structures. Using transmission electron microscopy (TEM) we observed that these structures corresponded to electron-dense double membrane vacuoles of different sizes and variable number. These vacuoles were positives for LysoTracker dye (retained in acidic subcellular compartments) and anti-LC3 immunofluorescence (used to monitor autophagy) revealing that, as was expected, these structures corresponded to autophagolysosomes. TEM images also revealed viral-like particles in some erythrocytes both in the cytoplasm and inside the autophagosomes. Therefore, Nkl could be involved in the autophagy process in erythrocytes as a defense mechanism against intracellular pathogens, such as viruses. Indeed, the hypothetical antiviral role of Nk-lysin is not fully understood. After a Viral Hemorrhagic Septicemia virus (VHSV) challenge, turbot nkl was overexpressed both in head kidney and spleen, suggesting again a certain function in the antiviral defense. Interestingly, the expression analysis of this gene in head kidney samples of VHSV-resistant and -susceptible selected families and the correlation between Nk-lysin expression in blood and susceptibility to VHSV, seem to indicate that higher basal levels of Nkl could induce higher resistance to the virus.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.

Abstract LB-233: Unique physicochemical properties of the potent FGFR 1, 2, 3 and 4 inhibitor JNJ-42756493 contribute to prolonged target shutdown

Abstract The ability of drugs to penetrate into cells and tissues is a fundamentally important property that is essential for the efficacy of drugs targeting intracellular processes. Many weakly basic small molecule drugs are known to accumulate extensively in acidic sub-cellular compartments such as lysosomes, through a process called ion trapping that is dependent on the physico-chemical properties of a compound. ‘Lysosomal uptake’ has been shown to significantly influence the activity of many agents, including anticancer drugs, both positively and negatively. Here we describe the unique physico-chemical properties of JNJ-42756493, a potent inhibitor of fibroblast growth factor receptors (FGFRs) 1, 2, 3 and 4, currently undergoing clinical testing for the treatment of malignancies with FGFR pathway activating alterations. JNJ-42756493 has weakly basic and lipophilic characteristics, is highly permeable, and is fluorescent when exposed to UV light. The fluorescent property of JNJ-42756493 enabled rapid and convenient visualization of the drug (spatial and temporal localization) within the lysosomes of cultured cells exposed to JNJ-42756493. In order to evaluate the influence of this lysosomotropic property on the activity of JNJ-42756493, FGFR phosphorylation was monitored in a series of wash-out experiments performed in various cellular models with FGFR1 or FGFR2 amplifications. This comparative washout analysis demonstrated prolonged FGFR kinase inhibition by JNJ-42756493 in comparison to other FGFR inhibitors lacking lysosomotropic properties. This qualitative fluorescence-based data was complemented by accurate quantification of the amount of compound taken up by the cells through the use of radiolabelled JNJ-42756493. Our results unequivocally demonstrate that JNJ-42756493 accumulates at high concentrations within the lysosomal compartment of cells without undergoing metabolism. The continued, long lasting effects on the target FGF receptor kinase inhibition following drug washout, suggest a novel intrinsic slow release of the unmodified drug from these intracellular stores. The lysosomotropic property of JNJ-42756493 is proposed to be a major contributor to the efficient and persistent inhibition of FGFR phosphorylation leading to potent cellular and in vivo activity. Citation Format: Eleonora Jovcheva, Caroline Paulussen, Patrick Van Bergen, Yolanda Chong, Jorge Vialard, Laurence Mevellec, Patrick Angibaud, Timothy Perera. Unique physicochemical properties of the potent FGFR 1, 2, 3 and 4 inhibitor JNJ-42756493 contribute to prolonged target shutdown. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-233. doi:10.1158/1538-7445.AM2014-LB-233

Multifunctional Tumor pH-Sensitive Self-Assembled Nanoparticles for Bimodal Imaging and Treatment of Resistant Heterogeneous Tumors

Nanoparticle-based diagnosis-therapy integrative systems represent an emerging approach to cancer treatment. However, the diagnostic sensitivity, treatment efficacy, and bioavailability of nanoparticles as well as the heterogeneity and drug resistance of tumors pose tremendous challenges for clinical implementation. We herein report on the fabrication of tumor pH-sensitive magnetic nanogrenades (termed PMNs) composed of self-assembled iron oxide nanoparticles and pH-responsive ligands. These PMNs can readily target tumors via surface-charge switching triggered by the acidic tumor microenvironment, and are further disassembled into a highly active state in acidic subcellular compartments that "turns on" MR contrast, fluorescence and photodynamic therapeutic activity. We successfully visualized small tumors implanted in mice via unique pH-responsive T1MR contrast and fluorescence, demonstrating early stage diagnosis of tumors without using any targeting agents. Furthermore, pH-triggered generation of singlet oxygen enabled pH-dependent photodynamic therapy to selectively kill cancer cells. In particular, we demonstrated the superior therapeutic efficacy of PMNs in highly heterogeneous drug-resistant tumors, showing a great potential for clinical applications.

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs(1,2,6,7). During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects(3-5). To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells. Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it(8-10). After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death. To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome(11-13). The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible(12,13). In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis(14), is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase. In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media(12,13). Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

Evidence for a non-replicative intracellular stage of nontypable Haemophilus influenzae in epithelial cells

Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi- or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells.

Trafficking microenvironmental pHs of polycationic gene vectors in drug-sensitive and multidrug-resistant MCF7 breast cancer cells

While multidrug resistance (MDR) has been a significant issue in cancer chemotherapy, delivery resistance to various anti-cancer biotherapeutics, including genes, has not been widely recognized as a property of MDR. This study aims to provide a better understanding of the transfection characteristics of drug-sensitive and drug-resistant cells by tracing microenvironmental pHs of two representative polymer vectors: poly( l-lysine) and polyethyleneimine. Drug-sensitive breast MCF7 cells had four- to seven-times higher polymeric transfection efficiencies than their counterpart drug-resistant MCF7/ADR-RES cells. Polyplexes in MCF7/ADR-RES cells after endocytosis were exposed to a more acidic microenvironment than those in MCF7 cells; the MDR cells show faster acidification rates in endosomes/lysosomes than the drug-sensitive cells after endocytosis (in the case of PLL/pDNA complexes, ∼ pH 5.1 for MCF7/ADR-RES cells vs. ∼ pH 6.8 for MCF7 cells at 0.5 h post-transfection). More polyplexes were identified trapped in acidic subcellular compartments of MCF7/ADR-RES cells than in MCF7 cells, suggesting that they lack endosomal escaping activity. These findings demonstrate that the design of polymer-based gene delivery therapeutics should take into account the pH of subcellular compartments.

Secretory state regulates Zn2+ transport in gastric parietal cell of the rabbit

Secretory compartments of neurons, endocrine cells, and exocrine glands are acidic and contain high levels of labile Zn2+. Previously, we reported evidence that acidity is regulated, in part, by the content of Zn2+ in the secretory [i.e., tubulovesicle (TV)] compartment of the acid-secreting gastric parietal cell. Here we report studies focusing on the mechanisms of Zn2+ transport by the TV compartment in the mammalian (rabbit) gastric parietal cell. Uptake of Zn2+ by isolated TV structures was monitored with a novel application of the fluorescent Zn2+ reporter N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ). Uptake was suppressed by removal of external ATP or blockade of H+-K+-ATPase that mediates luminal acid secretion. Uptake was diminished with dissipation of the proton gradient across the TV membrane, suggesting Zn2+/H+ antiport as the connection between Zn2+ uptake and acidity in the TV lumen. In isolated gastric glands loaded with the reporter fluozin-3, inhibition of H+-K+-ATPase arrested the flow of Zn(2+) from the cytoplasm to the TV compartment and secretory stimulation with forskolin enhanced vectorial movement of cytoplasmic Zn2+ into the tubulovesicle/lumen (TV/L) compartment. Our findings suggest that Zn2+ accumulation in the TV/L compartment is physiologically coupled to secretion of acid. These findings offer novel insight into mechanisms regulating Zn2+ homeostasis in the gastric parietal cell and potentially other cells in which acidic subcellular compartments serve signature functional roles.

The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER2

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.

Intracellular Activation of Trypsinogen in Rat Pancreatic Acini After Supramaximal Secretagogue Stimulation

The aim of the study was to explore the mechanism by which trypsinogen becomes activated during acute pancreatitis. Given the ability of cholecystokinin (CCK) to induce pancreatitis in vivo, the effects of high-dose CCK on preparations of isolated pancreatic acini were examined using immunofluorescence techniques for the detection of trypsinogen activation. Acini were pretreated with weak base or serine or cysteine protease inhibitors before CCK hyperstimulation. CCK was found to stimulate the generation of trypsinogen activation peptide (TAP), a marker for trypsinogen processing. The generation of TAP was inhibited by pretreatment with a weak base, chloroquine (40 micromol/L). TAP generation was also inhibited by pretreatment with serine protease inhibitor FUT-175 (1 micromol/L) but not cysteine protease inhibitor E64 (0.1 mmol/L) or E64-d (0.1 mmol/L). Although treatment with a high dose of E64-d (1 mmol/L) reduced activation, it also caused cell injury. High-dose CCK stimulated the intracellular activation of trypsinogen within isolated pancreatic acini. This event appears to require an acidic subcellular compartment and serine protease activity. The role for thiol proteases in this model remains unclear.

Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca(2+) flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7-6.7), whereas anti-TLR3-mediated Ca(2+) flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15-40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling.

Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine Red DND-99

Cationic amphiphilic drugs (CAD) containing a basic moiety often accumulate in lysosomes or other acidic subcellular compartments. This lysosomotropism is due to the protonation of the CAD within acidic organelles leading to the formation of a membrane-impermeable form. Highly lipophilic CADs show a greater propensity to accumulate than those with a lower lipophilicity (Log P). Here we describe a rapid method to quantitate the uptake of CAD within lysosomes. The principle of the method involves a competition between the CAD and the fluorescent basic amine probe Red DND-99 (LysoTracker) in HeLa cells. Visualization is performed using confocal fluorescence microscopy. Loading HeLa cells with 10–150 nM Red DND-99 gives a punctuated fluorescent pattern around the cell nucleus. This fluorescence is displaced by incubating the cells with 10–100 mM NH 4Cl, either before or after the addition of the probe, consistent with the reversible partitioning of the probe in the lysosome. The displacement of probe fluorescence by CAD such as chlorpromazine and imipramine was observed in a dose-dependent manner. Lower concentrations of CAD with high Log P displaced the probe more efficiently than those with a lower Log P. This assay represents a rapid and semiquantitative method for the measurement of lysosomotropism in an in vitro system without the use of radioactivity and cell fractionation. Furthermore, the microscopy confirms the targeting of the basic compounds to within the lysosomes.

Influence of Chloroquine on Diet-Induced Pancreatitis

Recent investigations have suggested that digestive zymogens may become activated within the acinar cell during acute pancreatitis. While the molecular events responsible for intracellular zymogen activation remain unknown, several potential enzymatic pathways require an acidic pH to optimally proceed. We therefore proposed that manipulation of subcellular pH might alter the course of experimental pancreatitis. Chloroquine, a weak base that raises the pH of acidic subcellular compartments, was administered to young female mice in which pancreatitis was induced by a choline-deficient, ethionine-supplemented (CDE) diet. Control animals were maintained on regular laboratory chow. Examination of isolated pancreatic acini using acridine orange cytofluorescence demonstrated expansion of acidic subcellular compartments in animals fed the CDE diet. These compartments were effectively neutralized in animals receiving chloroquine. Animals receiving continuous infusions of high-dose chloroquine demonstrated a significant (p < 0.05) decrease in free pancreatic tryptic activity as well as improved survival. These changes were also associated with decreased trypsinogen content in animals treated with high-dose chloroquine, suggesting an additional potential effect of chloroquine on zymogen synthesis and accumulation. One explanation of these findings is that a low-pH compartment may be important in the pathogenesis of diet-induced pancreatitis.

Posttranslational Processing of Defensins in Immature Human Myeloid Cells

AbstractHuman neutrophil promyelocytes synthesize, process, and package several microbicidal proteins, including the highly abundant defensins, into their azurophil granules. As deduced from their cDNA sequences, defensins are initially synthesized as 94 amino acid (aa) precursors that must undergo extensive processing. We performed metabolic labeling studies of defensin synthesis in the human promyelocytic cell line HL-60 and in chronic myeloid leukemia cells, and showed that preprodefensins are processed to mature 29 to 30 aa defensins over 4 to 24 hours via two major intermediates: a 75 aa prodefensin generated by the cleavage of the signal sequence, and a 56 aa prodefensin that results from a subsequent preaspartate proteolytic cleavage. Almost all of the 75 aa form was found in the cytoplasmic/microsomal fraction, whereas the 56 aa prodefensin and mature defen-sins predominated in the granule-enriched fraction. The 75 aa prodefensin was also selectively released into the culture supernatant. Treatment of HL-60 cells with monensin, chloro-quin, or ammonium chloride, substances that neutralize acidic subcellular compartments, partially blocked conversion of the 75 aa prodefensin into 56 aa prodefensin, but did not increase the extracellular release of the 75 aa form. Further studies will be required to determine the role of this processing pathway in subcellular targeting to azurophil granules and avoidance of autocytotoxicity. © 1992 by The American Society of Hematology.