Lysis Of Lysosomes Research Articles

Abstract 6020: Preclinical activity of LM-305 targeting G-protein-coupled receptor class 5 member D (GPRC5D) antibody drug conjugate for the treatment of multiple myeloma

Abstract Emerging therapies including chimeric antigen receptor (CAR) T therapy targeting B cell maturation antigen (BCMA) have shown promising clinical outcomes in relapsed and refractory multiple myeloma (RRMM). However, majority of patients eventually develop progressive disease due to heterogeneity in BCMA expression or loss of antigen during treatment. Therefore, new targets are required to overcome the problem of resistance encountered with these agents. The expression of GPRC5D (G-protein-coupled receptor class 5 member D) is high on MM cells and low in normal tissues. Although high levels of GPRC5D and BCMA were found in similar proportions of MM patients, they are independently expressed by MM cells. GPRC5DxCD3 bispecific T-cell-redirecting antibody is currently under clinical investigation. Preliminary results have shown early evidence in efficacy with tolerable safety profile. These findings identified GPRC5D as an ideal therapeutic antibody drug conjugate (ADC) target for the treatment of patients with RRMM. Therefore, the present study was aimed at developing a novel anti-GPRC5D ADC drug LM-305 and evaluating its preliminary efficacy by performing preclinical activity using in vitro and in vivo mouse models. The binding affinity of LM-305 was evaluated by flow cytometry in engineered GPRC5D over-expressing MM tumor cells and endogenous GPRC5D-expressing MM tumor cells. The internalization of LM-305 was assessed with pH-dependent dye in NCI-H929 and MM.1R cell lines. The cytotoxicity of LM-305 was assessed by cell viability assay in MM tumor cell lines. In vivo anti-tumor activity of multiple doses of LM-305 (1mg/kg, 3mg/kg, 10mg/kg) was evaluated in NCI-H929 and MM.1R tumor cell line derived xenograft (CDX) models. In vitro studies revealed that LM-305 binds with high affinity to GPRC5D over-expressing cell lines and GPRC5D endogenously expressing MM cells in a dose-dependent manner. It can be efficiently internalized by GPRC5D expressing cells and further lysosomal lysis was detected by pHrodo dye. LM-305 displayed potent cytotoxicity when co-cultured with MM tumor cells (NCI-H929 and MM.1R) with IC50 values ranging from 0.1 to 0.3 nM. In vivo tumor xenograft models suggested that treatment with LM-305 resulted in dose-dependent inhibition of tumor growth in tumor bearing mice. Additionally, LM-305 exhibited complete response (CR) in the GPRC5D high-expressing MM CDX models at a dose of 3mg/kg. Moreover, LM-305 showed good safety profile in the animal studies. In conclusion, this preclinical data suggested that LM-305 is a novel GPRC5D targeting ADC with best-in-class potential, and therefore it can be a promising therapeutic candidate for the treatment of RRMM patients expressing GPRC5D. Citation Format: Wentao Huang, Jie Luo, Yifan Li, Da Fei, Xia Qin, Runsheng Li. Preclinical activity of LM-305 targeting G-protein-coupled receptor class 5 member D (GPRC5D) antibody drug conjugate for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6020.

The butyrophilin 1a1 knockout mouse revisited: Ablation of Btn1a1 leads to concurrent cell death and renewal in the mammary epithelium during lactation.

Butyrophilin 1A1 (BTN1A1) is implicated in the secretion of lipid droplets from mammary epithelial cells as a membrane receptor, which forms a secretion complex with the redox enzyme, xanthine oxidoreductase (XDH). The first evidence that BTN1A1 functions in this process was the generation of Btn1a1 −/− mouse lines, in which lipid secretion was disrupted and large unstable droplets were released into alveolar spaces with fragmented surface membranes. We have revisited one of these mutant mouse lines using RNAseq and proteomic analysis to assess the consequences of ablating the Btn1a1 gene on the expression of other genes and proteins. Disruption of intact Btn1a1 protein expression led to a large build‐up of Xdh in the cytoplasm, induction of acute phase response genes and Lif‐activation of Stat3 phosphorylation. At peak lactation, approx. 10% of the cells were dying, as assessed by TUNEL‐analysis of nuclear DNA. Possible cell death pathways included expression of caspase 8 and activated caspase 3, autophagy, Slc5a8‐mediated inactivation of survivin (Birc5), and pStat3‐mediated lysosomal lysis, the latter of which is the principal death route in involuting wild type cells. Milk secretion was prolonged by renewal of the secretory epithelium, as evidenced by the upregulation of Ki67 in approx. 10% of cell nuclei and expression of cyclins and Fos/Jun. These data highlight the plasticity of the mammary epithelium and the importance of functional BTN1A1 expression for maintenance of terminally differentiated secretory cells and optimal milk production throughout lactation.

Bioinspired Design of Lysolytic Triterpenoid-Peptide Conjugates that Kill African Trypanosomes.

Humans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). The active TLF ingredient is the primate-specific apolipoprotein L1 (APOL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies, only two are able to counteract the activity of APOL1; this illustrates its evolutionarily optimized design and trypanocidal potency. Herein, we ask whether a synthetic (syn) TLF can be synthesized by using the design principles of the natural TLF complexes but with different chemical building blocks. We demonstrate the stepwise development of triterpenoid-peptide conjugates, in which the triterpenoids act as a cell-binding, uptake and lysosomal-transport modules and the synthetic peptide GALA acts as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis thus demonstrating a proof-of-principle for the bioinspired, forward-design of a synTLF.

Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B’s proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis.

Abstract A57: miR-125a-induced cellular switch elicits a response to anti-HER2 targeted therapy in breast cancer cells

Abstract Background: The EGFR/HER2 signaling network emerges as an effective therapeutic target for Her2 enriched cancers, which is known by its aggressive biological course. Nevertheless, there is evidence that the EGFR/HER2 network may play a key role not only in the HER2-enriched subtype of breast cancer, but also in the similarly aggressive basal-like subtype. We had formerly demonstrated the involvement of miR-125a-3p in the EGFR pathway in prostate cancer. We aimed to study the effect of miR-125a-3p as a potential modulator of the ERBB2/HER2 pathway in basal-like breast cancer subtype. Methods: Using qPCR we calibrated estrogen receptor (ER), ERBB2 (HER2), miR-125a-3p/5p (two isoforms) expression in three cell lines: MDA-MB-231, MCF-7 and SKBR3 (represent the basal-like subtype, ER positive subtype and HER2-enriched subtype respectively). We generated stable MDA-MB-231 cells that overexpress miR-125a-3p and control cells that overexpress scrambled miRNA. In parallel, we established an in vivo platform by injection of miR-125a-3p-overexpressing MDA-MB-231 breast cancer cells to the mammary pad of NUDE mice and evaluated traits of the induced tumors compared with scrambled miRNA-expressing cells (control mice) and response to potential targeted therapy. Results: miR-125a-3p was endogenously expressed in all cell lines, though its expression in MDA-MB-231 cells was significantly lower than in MCF-7 or in SKBR3 cells. Following transfection of cells with miR-125a-3p, MDA-MB-231 cells showed a significant increase in the expression level of ERBB2 mRNA and protein as well as a stronger immunofluorescence staining of ErbB2 than in control cells. Combined treatment of miR-125a-3p-overexpressing cells and trastuzumab induces apoptosis and reduces migration of MDA-MB-231 cells. By tracking Erbb2 internalization and its localization to the lysosoms/the functionality of erbb2 receptor, we determined that trastuzumab caused internalization of ErbB2 and a subsequent lysosomal lysis. In the experimental in vivo platform, starting one week post cells injection/tumor formation?, mice were treated twice a week, for the next 28 days, with 10 mg/Kg trastuzumab or saline as a control. Response to treatment was evaluated by measurement of the tumor size/volume by computerized tomography (CT) or calliper. Both measurements indicated that that the tumors in the miR-125a-3p group that were treated with trastuzumab were significantly smaller than in other groups. Conclusion: our results indicate that miR-125a-3p is capable of inducing a shift in the involvement of key cellular pathway - the ERBB2 pathway that may convert the cell fate and dispose it to anti-HER2 therapies. In an era of personalized medicine, our study proposes a means to enlarge the patient population that may benefit from anti-HER2 therapies. Citation Format: Lihi Ninio-Many, Elad Hikri, Salomon M. Stemmer, Ruth Shalgi, Irit Ben-Aharon. miR-125a-induced cellular switch elicits a response to anti-HER2 targeted therapy in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A57.

Tuning the Buffering Capacity of Polyethylenimine with Glycerol Molecules for Efficient Gene Delivery: Staying In or Out of the Endosomes

Endosomal escape is a major bottleneck for efficient non-viral gene delivery. This paper presents the development of two novel non-viral vectors by cross-linking glycerol molecules with low molecular weight polyethylenimine (PEI). The vectors, namely, HG-PEI (45 mol% glycerol content) and LG-PEI (9 mol% glycerol content) have apparently similar DNA binding, DNA unpacking and cellular uptake abilities but differ in buffering capacity. The cellular uptake and subsequent transfection efficiency of LG-PEI is superior to commercially available PEI 25 k. Interestingly, although the cellular uptake of HG-PEI is higher than that of PEI 25 k, the transgene expression by HG-PEI-mediated transfection is very low. Inhibitor and co-localization studies demonstrate the mechanism of endocytosis and formation of endosomes prone to lysosomal lysis of HG-PEI polyplexes as a consequence of its weak buffering capacity. Importantly, when the lysosomal lysis is inhibited, the transgene expression of HG-PEI-mediated transfection increases by 9-fold of its initial capacity which is comparable to the transfection efficiency of PEI 25 k. These results indicated that the buffering capacity of the polymers primarily impacts endosomal escape and subsequent transfection efficiency. Furthermore, this study highlights the significance of cross-linkers in optimizing the buffering capacity when designing polymers for gene delivery.

Overcoming Multidrug Resistance via Photodestruction of ABCG2-Rich Extracellular Vesicles Sequestering Photosensitive Chemotherapeutics

Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing lysosomal photodestruction of normal breast epithelial cells. Thus, MDR modalities including ABCG2-dependent drug sequestration within EVs can be rationally converted to a pharmacologically lethal Trojan horse to selectively eradicate MDR cancer cells.

Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization

Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 min after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.

Regulated ATP release from astrocytes through lysosome exocytosis

Release of ATP from astrocytes is required for Ca2+ wave propagation among astrocytes and for feedback modulation of synaptic functions. However, the mechanism of ATP release and the source of ATP in astrocytes are still not known. Here we show that incubation of astrocytes with FM dyes leads to selective labelling of lysosomes. Time-lapse confocal imaging of FM dye-labelled fluorescent puncta, together with extracellular quenching and total-internal-reflection fluorescence microscopy (TIRFM), demonstrated directly that extracellular ATP or glutamate induced partial exocytosis of lysosomes, whereas an ischaemic insult with potassium cyanide induced both partial and full exocytosis of these organelles. We found that lysosomes contain abundant ATP, which could be released in a stimulus-dependent manner. Selective lysis of lysosomes abolished both ATP release and Ca2+ wave propagation among astrocytes, implicating physiological and pathological functions of regulated lysosome exocytosis in these cells.

Enhancement of Lysosomal Osmotic Sensitivity Induced by the Photooxidation of Membrane Thiol Groups¶

The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5′-dithio-bis(2-nitrobenzoic acid) and examining the lysosomal β-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue–mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75 000 to 125 000), which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis.

NAADP Mobilizes Calcium from the Endoplasmic Reticular Ca2+ Store in T-lymphocytes

The target calcium store of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent endogenous calcium-mobilizing compound known to date, has been proposed to reside in the lysosomal compartment or in the endo/sarcoplasmic reticulum. This study was performed to test the hypothesis of a lysosomal versus an endoplasmic reticular calcium store sensitive to NAADP in T-lymphocytes. Pretreatment of intact Jurkat T cells with glycyl-phenylalanine 2-naphthylamide largely reduced staining of lysosomes by LysoTracker Red and abolished NAADP-induced Ca(2+) signaling. However, the inhibitory effect was not specific since Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate and cyclic ADP-ribose was abolished, too. Bafilomycin A1, an inhibitor of the lysosomal H(+)-ATPase, did not block or reduce NAADP-induced Ca(2+) signaling, although it effectively prevented labeling of lysosomes by LysoTracker Red. Further, previous T cell receptor/CD3 stimulation in the presence of bafilomycin A1, assumed to block refilling of lysosomal Ca(2+) stores, did not antagonize subsequent NAADP-induced Ca(2+) signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca(2+) signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca(2+) signaling was obtained. The sensitivity of NAADP-induced Ca(2+) signaling toward thapsigargin, combined with our recent results identifying ryanodine receptors as the target calcium channel of NAADP (Dammermann, W., and Guse, A. H. (2005) J. Biol. Chem. 280, 21394-21399), rather suggest that the target calcium store of NAADP in T cells is the endoplasmic reticulum.

Inhibition of weak-base amine-induced lysis of lysosomes by cytosol.

Certain amines known to be concentrated in lysosomes, termed "lysosomotropic amines," cause the formation of lysosomal vacuoles. A cell-free system was established to examine the effects of basic substances and acidic ionophores. In this system, the drugs not only increased the internal pH, but also caused a disruption of lysosomes. The osmotic swelling of lysosomes induced by protonated bases or cations for particular ionophores, which had accumulated within lysosomes driven by the proton pump, caused the osmotic lysis of lysosomes. The lysosomal disruption was inhibited upon the addition of the cytosol fraction. This phenomenon provides an in vitro system for studying the osmo-regulation and intercellular dynamics of the lysosomal system, including membrane fusion. The lysosomal stabilization factor was purified from the cytosol fraction and identified as ATP-stimulated glucocorticoid receptor translocation promoter (ASTP).

GTP gamma S-stimulated lysosomal lysis dependent on the assembly of adaptor protein on lysosome.

Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) destroys the dextran-loaded lysosomes (J. Biochem., 123, 637 (1998)). The transfer of the ADP-ribosylation factor (ARF) from the cytosol to the lysosomal membrane is necessary for this lysis to occur. The role of ARF in the biogenesis of the Golgi complex is to generate high-affinity membrane-binding sites for the heterotetrameric adaptor protein complex in Golgi membranes. We have found that ARF also recruits the adaptor protein to lysosomes. The recruitment of protein coats for vesicles is necessary for the GTPgammaS-stimulated lysis of lysosomes. The GTPgammaS-induced lysis proceeded via a process similar to that for the assembly of coated proteins to coated vesicles, which serve to transport proteins between intracellular organelles.

Enhancement of lysosomal osmotic sensitivity induced by the photooxidation of membrane thiol groups.

The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) and examining the lysosomal beta-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75000 to 125000), which was visualized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis.

Acid Phosphatase Activity in Human Breast Tumors

We examined lysosomal participation in the degradation of tumor cells from human breast biopsies, utilizing the histochemical activity and localization of acid phosphatase (AP). Enzyme activities in benign and malignant lesions were compared. AP was faintly detected in normal mammary epithelia and was marked in malignant cells. The histochemical patterns of AP distribution in the breast tissues showed differences between normal and neoplastic cells. AP staining was more intense in ductal carcinoma in situ (DCIS) associated with invasive ductal carcinoma (IDC) cases compared to when IDC was present alone. These results suggest that non-invasive breast cancers, such as DCIS, may be subjected to more lysosomal cellular lysis than is observed in invasive breast cancers, like IDC.

A rapid method to separate endosomes from lysosomal contents using differential centrifugation and hypotonic lysis of lysosomes

Here we describe a fast and efficient subcellular fractionation procedure that permits lysosomes to be separated from endosomes. Differential centrifugation is used to isolate a subcellular fraction containing both endosomes and lysosomes. Because lysosomes are sensitive to osmotic stress, hypotonic conditions destroy them, whereas endosomes, which are osmotically insensitive, stay intact. We demonstrate that hypotonic lysis of an endosome–lysosome-pool releases 85% of the lysosomes into the supernatant as measured by the activity of the lysosomal marker enzyme N-acetyl-β- d-glucosaminidase (β-AGA). The endosomal fraction is thoroughly characterised using a variety of subcellular markers. After pulsing cells with fluorescein isothiocyanate labelled transferrin (FITC-Tf), only about 12% of the marker is released under hypotonic conditions. A typical fractionation procedure takes about 1–2 h from initial cell homogenisation. The fractionation gives a pure lysosomal fraction (fraction L) containing high activities of lysosomal enzymes and an endosomal fraction (fraction E) reflecting different stages of endosomes.

ARF-induced lysosomal lysis in vitro.

Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) disintegrated lysosomes in a dose-dependent manner, as detected as the release of preloaded fluorescein isothiocyanate-dextran. The effect of GTPgammaS was suppressed by GTP or GDP, indicating a role of a GTP binding protein (G-protein) in the lysis [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877]. Gel filtration of cytosol and GTP-ligand blotting showed that a small GTP-binding protein participated in the lysosomal lysis. We partially purified the G-protein from rat liver cytosol and identified it as ARF1. GTPgammaS-stimulated lysis was reconstituted with ARF1 purified from bovine brain cytosol or recombinant ARF1. ARF bound to lysosomal membranes depending upon GTPgammaS in a dose-dependent manner. These results suggest that the transfer of ARF from the cytosol to the lysosomal membrane is necessary for GTPgammaS-stimulated lysis of lysosomes.

Disintegration of lysosomes mediated by GTPgammaS-treated cytosol: possible involvement of phospholipases.

We showed previously that cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS) disintegrated lysosomes in vitro [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877] in time-, temperature-, and dose-dependent manners. This also requires ATP, however, the latter can be substituted with deoxy-ATP, ADP, or ATPgammaS, suggesting no requirement of ATP hydrolysis. The lysis was inhibited by several chemical modifiers, including N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and by various phospholipase inhibitors (trifluoperazine, p-bromophenacyl bromide, nordihydroguaiaretic acid, W-7, primaquine, compound 48/80, neomycin, and gentamicin), but not by ONO-RS-082, an inhibitor of phospholipase A2. The reaction was also inhibited by phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylcholine) and diacylglycerol. Among the phospholipase A2 hydrolysis products of phospholipids, unsaturated fatty acids (oleate, linoleate, and arachidonate) and lysophospholipid (lysophosphatidylcholine) by themselves broke lysosomes down directly, whereas saturated fatty acids (palmitate and stearate) had little effect. We found that GTPgammaS-stimulated cytosolic phospholipase A2 activity was highly sensitive to ONO-RS-082. These results suggest the participation of phospholipase(s), though not cytosolic phospholipase A2, in the GTPgammaS-dependent lysis of lysosomes.

The direct cause of photodamage-induced lysosomal destabilization

Whether membrane lipid photoperoxidation is the immediate cause for lysosomal lysis is still unclear. In this study, we investigated the direct causal factor of photoinduced lysosomal destabilization in a K +-containing solution. Methylene blue (MB)-mediated photodamage caused lysosomal membrane lipid peroxidation and loss of membrane fluidity. Compared with unirradiated lysosomes, the photodamaged lysosomes significantly lost enzyme latency in an isotonic K +-containing solution during a 20-min period of incubation. It indicates an increase in lysosomal K + permeability. The inward K + permeation of photodamaged lysosomes was further proved by a K +-induced elevation of internal membrane potential. In addition, the photodamaged lysosomes displayed an increased osmotic sensitivity, showing that MB-mediated photodamage promotes lysosomal osmotic fragility. Although these photoinduced alterations occurred, the lysosomes were relatively stable in an isotonic sucrose medium. In contrast, the organelle destabilized in a photodamage-dependent fashion in an isotonic K +-containing solution. The results indicate that membrane lipid peroxidation does not definitely destabilize lysosomes. The direct cause for the lysosomal destabilization is photoinduced osmotic imbalance across its membrane via an increased K + uptake, while the increase in osmotic sensitivity favors the destabilization of photodamaged lysosomes.

Thyroid Hormone Residues Are Released from Thyroglobulin with Only Limited Alteration of the Thyroglobulin Structure

We have tried to characterize thyroglobulin (Tg) degradation products in purified pig thyroid lysosomes to determine whether the release of thyroid hormone residues from Tg involves a random proteolytic attack or discrete and selective cleavage reactions. The intralysosomal soluble protein fraction was prepared by osmotic pressure-dependent lysis of lysosomes purified by isopycnic centrifugation on Percoll gradients. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a fraction of Tg (5-10% of total lysosomal protein) with the same molecular weight as that of the intact Tg subunit. This high molecular weight Tg was the only intralysosomal species detected by Western blot using antipig Tg antibodies. In nondenaturing conditions, lysosomal Tg (LTg) identified by radioimmunoassay was in the form of a dimer with a sedimentation coefficient lower than that of either iodinated Tg (colloid Tg) or noniodinated Tg (microsomal Tg). LTg had a lower iodine content than colloid Tg:9-12 versus 39-42 iodine atoms/molecule. Pronase hydrolysates of LTg did not contain any 3,5,3',5'-tetraiodo-L-thyronine or 3,3',5-triiodo-L-thyronine residues detectable by reverse-phase high pressure liquid chromatography; iodine present in LTg was in the form of iodotyrosines. Under reducing conditions, LTg almost completely disappeared and gave rise to various polypeptides of smaller size. These results suggest that Tg transferred to lysosomes is subjected to selective proteolytic cleavage reaction(s) that release thyroid hormone residues. This early step would lead to the formation of hormone-depleted Tg molecules that are cleaved at discrete sites, the resulting polypeptides remaining bound through disulfide bonds to yield Tg molecules with an apparently normal size and a slightly altered structure.