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Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells
Abstract
Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafilomycin A1 is a valuable tool for distinguishing among the three different types of ATPases and represents the first relatively specific potent inhibitor of vacuolar ATPases.
... Quina treatment blocked the cytosolic release of His_DTA as indicated by hampered cell rounding (Figure 2a,b) and diminished loss of cell viability (Figure 2c) compared to cells treated only with the combination His_DTA/C2IIa. Moreover, even stronger inhibition of His_DTA/C2IIa-mediated toxicity was detected when using Bafilomycin A1 (BafA1), a well-established inhibitor of endosomal acidification [5,48] (Figure 2a-c). These data indicate that the cytosolic delivery of His-tagged protein cargo via C2IIa is dependent on endosomal acidification and translocation through the C2IIa pore. ...
... Already after 30 min of incubation, His_eGFP_DTA was internalized when HeLa cells were treated with the combination His_eGFP_DTA/C2IIa (Figure 2d). inhibitor of endosomal acidification [5,48] (Figure 2a-c). These data indicate that the cytosolic delivery of His-tagged protein cargo via C2IIa is dependent on endosomal acidification and translocation through the C2IIa pore. ...
... Moreover, for uptake and translocation of the His-tagged cargo proteins, the mode of action of C2IIa is exploited. The delivery of the His-tagged proteins via C2IIa was successfully inhibited by the V-ATPase inhibitor BafA1 [5,48] and the C2IIa pore blocker Quina [46,47], demonstrating the dependency on endosomal acidification and protein translocation through the C2IIa pore. Schleberger et al. suggested that under low pH conditions in the endosome, the N-terminal Helix α1 of C2I possesses a positive net charge and could form a bolt that initiates translocation through the C2IIa pore [13]. ...
The binary Clostridium (C.) botulinum C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and translocate the enzyme subunit C2I into the cytosol of target cells. Here, we investigate whether C2IIa can be harnessed as a transporter for proteins/enzymes fused to polycationic tags, as earlier demonstrated for the related anthrax toxin transport subunit PA63. To test C2IIa-mediated transport in cultured cells, reporter enzymes are generated by fusing different polycationic tags to the N- or C-terminus of other bacterial toxins’ catalytic A subunits. C2IIa as well as PA63 deliver N-terminally polyhistidine-tagged proteins more efficiently compared to C-terminally tagged ones. However, in contrast to PA63, C2IIa does not efficiently deliver polylysine-tagged proteins into the cytosol of target cells. Moreover, untagged enzymes with a native cationic N-terminus are efficiently transported by both C2IIa and PA63. In conclusion, the C2IIa-transporter serves as a transport system for enzymes that harbor positively charged amino acids at their N-terminus. The charge distribution at the N-terminus of cargo proteins and their ability to unfold in the endosome and subsequently refold in the cytosol determine transport feasibility and efficiency.
... To prevent loading of acidic Ca 2+ stores, the macrolide antibiotic bafilomycin A1 (Baf-A1), an inhibitor of the lysosomal vacuolar-type H + -ATPase (V-ATPase) was used in many cell types. The V-ATPase plays a role in acidifying the lumen of endo-lysosomes (Bowman et al. 1988;Dröse and Altendorf 1997;Huss et al. 2011), and Baf-A1 can inhibit V-ATPase with high affinity at nanomolar concentrations (Bowman et al. 1988). The idea is that inhibiting the H + uptake into the lumen via the V-ATPase leads to passive loss of H + through leaks, which in turn dissipates the H + gradient across the lysosomal membrane and causes the lumen to become alkaline. ...
... To prevent loading of acidic Ca 2+ stores, the macrolide antibiotic bafilomycin A1 (Baf-A1), an inhibitor of the lysosomal vacuolar-type H + -ATPase (V-ATPase) was used in many cell types. The V-ATPase plays a role in acidifying the lumen of endo-lysosomes (Bowman et al. 1988;Dröse and Altendorf 1997;Huss et al. 2011), and Baf-A1 can inhibit V-ATPase with high affinity at nanomolar concentrations (Bowman et al. 1988). The idea is that inhibiting the H + uptake into the lumen via the V-ATPase leads to passive loss of H + through leaks, which in turn dissipates the H + gradient across the lysosomal membrane and causes the lumen to become alkaline. ...
... The pH stability could be evaluated using lysotracker or other more specific pH sensors. It was argued that ML-SA1 application induced Ca 2+ release from the lysosome lumen in the Fura-Dextran-loaded ML1-mCherry-transfected HEK-293T cells, since the LysoTracker staining was not significantly reduced by ML-SA1, suggesting that the signals were primarily mediated by changes of intralysosomal Ca 2+ , but not by changes in the intralysosomal pH (Garrity et al. 2016 (Thastrup et al. 1990;Yagodin et al. 1999) GPN ES/LYS (Berg et al. 1994;Haller et al. 1996;Churchill et al. 2002) Bafilomycin A1 ES/LYS V-ATPase (Bowman et al. 1988 ...
Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca2+-permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca2+ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca2+ measurements, (2) peri-endo-lysosomal Ca2+ imaging using genetically encoded Ca2+ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca2+ imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca2+ imaging by directing Ca2+ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca2+ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca2+ imaging.
... treatment with sub-lethal levels of bafilomycin A1 (BafA1), an inhibitor of the membrane-localized vacuolar-type proton ATPase (V-type ATPase) that interferes with endosomal trafficking. 55 This multi-subunit complex is responsible for the acidification of intracellular vacuoles, including lysosomes. Cotreatment of trypanosomes conferred a 5−7 fold increase in resistance to 9a ( Figure 6A), a finding that could be correlated with a general reduction in uptake ( Figure 6B). ...
... We also found that uptake and trafficking to the lysosome could be inhibited by BafA1 ( Figure 6). This macrolide antibiotic inhibits V-type ATPase-mediated endosomal acidification, 55 affecting both fluid phase and receptor-mediated endocytosis and blocking the maturation pathway of early to late endosomes. 64,65 Although BafA1 is also a potassium ionophore and inhibits mitochondrial function, 66,67 this only occurs at levels 20 times higher than the BafA1 concentrations used in the current study ( Figure 6). ...
We designed and synthesized a series of symmetric bis-6-amidino-benzothiazole derivatives with aliphatic central units and evaluated their efficacy against bloodstream forms of the African trypanosome Trypanosoma brucei. Of these, a dicationic benzothiazole compound (9a) exhibited sub-nanomolar in vitro potency with remarkable selectivity over mammalian cells (>26,000-fold). Unsubstituted 5-amidine groups and a cyclohexyl spacer were the crucial determinants of trypanocidal activity. In all cases, mice treated with a single dose of 20 mg kg–1 were cured of stage 1 trypanosomiasis. The compound displayed a favorable in vitro ADME profile, with the exception of low membrane permeability. However, we found evidence that uptake by T. brucei is mediated by endocytosis, a process that results in lysosomal sequestration. The compound was also active in low nanomolar concentrations against cultured asexual forms of the malaria parasite Plasmodium falciparum. Therefore, 9a has exquisite cross-species efficacy and represents a lead compound with considerable therapeutic potential.
... Ot is a vector-borne pathogen that is transmitted by the larval stage of trombiculid mites and causes potentially lethal febrile infection in humans (Luce-Fedrow et al., 2018). About one million cases of scrub typhus are estimated annually by the World Health Organization, mainly in Southeast Asia (Bonell et al., 2017). In its vector mites, it is maintained as an endosymbiont by transovarian and transstadial transmission (Rapmund et al., 1969;Sonthayanon et al., 2010;Takhampunya et al., 2016). ...
... of the vacuolar H + -ATPase that blocks lysosome acidification (Bowman et al., 1988), it was observed that a low endosomal pH is required for the escape of Ot to the cytosol and subsequent cytosolic replication (Chu et al., 2006). While mechanistic details on the contribution of host factors to the escape of Ot are still lacking, it is known from infection of epithelial cells by Shigella flexneri that the invasion does not only involve the formation of a bacteria-containing vacuole but also triggers the recruitment of further cellular factors, for example, macropinosomes and small Rab GTPases (Weiner et al., 2016). ...
The life cycle of the mite‐borne, obligate intracellular pathogen Orientia tsutsugamushi (Ot), the causative agent of human scrub typhus, differs in many aspects from that of other members of the Rickettsiales order. Particularly, the nonlytic cellular exit of individual Ot bacteria at the plasma membrane closely resembles the budding of enveloped viruses but has only been rudimentarily studied at the molecular level. This brief article is focused on the current state of knowledge of escape events in the life cycle of Ot and highlights differences in strategies of other rickettsiae. image
... Bafilomycins (including the A1, B, C and D forms) are macrolide antibiotics [20][21][22][23][24][25][26][27] that inhibit V-type ATPases [23,28]. Bafilomycin A1 thereby prevents the fusion between lysosomes and autophagosomes [20,24,28,29]. ...
... Bafilomycins (including the A1, B, C and D forms) are macrolide antibiotics [20][21][22][23][24][25][26][27] that inhibit V-type ATPases [23,28]. Bafilomycin A1 thereby prevents the fusion between lysosomes and autophagosomes [20,24,28,29]. ...
Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.
... Next, we investigated the impact of impairing the lysosomal degradative pathway on AKH-αR5W4 RhodB cell penetration and traffic. Bafilomycin A1 (BafA1) is a chemical compound in the macrolide antibiotic family, produced from a variety of streptomycetes, which acts as a specific inhibitor of vacuolar H + ATPase (V-ATPase), an enzyme responsible for the acidification of cellular compartments such as endosomes, lysosomes and secretory vesicles (Bowman et al., 1988). By blocking V-ATPase function, BafA1 disrupts intracellular acidification by inhibiting the flow of protons across membranes, leading to an increase in pH in intracellular compartments (Wang et al., 2021). ...
CPPs, or Cell-Penetrating Peptides, offer invaluable utility in disease treatment due to their ability to transport various therapeutic molecules across cellular membranes. Their unique characteristics, such as biocompatibility and low immunogenicity, make them ideal candidates for delivering drugs, genes, or imaging agents directly into cells. This targeted delivery enhances treatment efficacy while minimizing systemic side effects. CPPs exhibit versatility, crossing biological barriers and reaching intracellular targets that conventional drugs struggle to access. This capability holds promise in treating a wide array of diseases, including cancer, neurodegenerative disorders, and infectious diseases, offering a potent avenue for innovative and targeted therapies, yet their precise mechanism of cell entry is far from being fully understood. In order to correct Cu dysregulation found in various pathologies such as Alzheimer disease, we have recently conceived a peptide Cu(II) shuttle, based on the αR5W4 CPP, which, when bound to Cu(II), is able to readily enter a neurosecretory cell model, and release bioavailable Cu in cells. Furthermore, this shuttle has the capacity to protect cells in culture against oxidative stress-induced damage which occurs when Cu binds to the Aβ peptide. The aim of this study was therefore to characterize the cell entry route used by this shuttle and determine in which compartment Cu is released. Pharmacological treatments, siRNA silencing and colocalization experiments with GFP-Rab fusion proteins, indicate that the shuttle is internalized by an ATP-dependent endocytosis pathway involving both Rab5 and Rab14 endosomes route and suggest an early release of Cu from the shuttle.
... The incubation medium contained cell samples, 5 mM ATP, 5 mM MgSO 4 , and buffer solutions according to pH optima for three major yeast ATPases: 20 mM MES−NaOH buffer, pH 6.5 for the cell membrane ATPase; 50 mM Tris-HCl, pH 7.0 for the vacuolar membrane ATPase; and 50 mM Tris-HCl, pH 8.5 for the mitochondrial ATPase. Activity of these ATPases was determined using specific inhibitors: 100 μM Na 3 VO 4 ·12H 2 O as an inhibitor of the cell membrane ATPase (Borst-Pauwels and Peters, 1981); 0.5 μM bafilomycin A (Sigma-Aldrich, United States) as an inhibitor of the vacuolar membrane ATPase (Bowman et al., 1988); and 5 mM NaN 3 as an inhibitor of the mitochondrial ATPase (Harris, 1989). In all three cases, ATP hydrolysis was carried out at 30°C. ...
The Ррх1 exopolyphosphatase of yeast is a constitutive protein localized predominantly in the cytoplasm. The purified enzyme hydrolyzes inorganic polyphosphates with high activity; however, in the knockout ∆ppx1 mutant of Saccharomyces cerevisiae the increase in the polyphosphate level was small, and no changes in physiological properties of this mutant were observed. To elucidate the functions of Ppx1, we studied the physiological characteristics of the S. cerevisiae strain overexpressing this enzyme. When cultivated in the YPD medium, the strain overexpressing Ppx1 showed no growth features different from those of the parental strain. The following physiological features of the strain overexpressing Ppx1 were observed at the stationary stage of growth: the level of ATP increased by nine times, the activity of vacuolar ATPase significantly decreased, and the sensitivity to peroxide increased compared to the parental strain. The level of reactive oxygen species doubled, while the degree of lipid oxidation remained the same as in parental strain. Since overexpression of Ppx1 under the culture conditions used did not affect the polyphosphate level, these polymers were not the regulators of the changes described above. Response to oxidative stress and vacuolar ATPase activity in yeasts is known to be regulated by cAMP, while Ppx1 is capable of hydrolyzing this signaling compound. We suggest that one of the functions of Ppx1 in yeasts is participation in the regulation of cAMP level.
... In contrast to eukaryotes, specific V-type ATPase inhibitors were not described for prokaryotes yet. In eukaryotic cells, bafilomycin A and archazolid A were shown to act as V-type ATPase inhibitors [45][46][47]. We tested these compounds in growth assays up to a concentration of 10 µM on E. faecalis but could not find any inhibitory effect (data not shown). ...
Background
All gastrointestinal pathogens, including Enterococcus faecalis and Enterococcus faecium, undergo adaptation processes during colonization and infection. In this study, we investigated by data-independent acquisition mass spectrometry (DIA-MS) two crucial adaptations of these two Enterococcus species at the proteome level. Firstly, we examined the adjustments to cope with bile acid concentrations at 0.05% that the pathogens encounter during a potential gallbladder infection. Therefore, we chose the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA), as these are the most prominent bile acids. Secondly, we investigated the adaptations from an aerobic to a microaerophilic environment, as encountered after oral-fecal infection, in the absence and presence of deoxycholic acid (DCA).
Results
Our findings showed similarities, but also species-specific variations in the response to the different bile acids. Both Enterococcus species showed an IC50 in the range of 0.01- 0.023% for DCA and CDCA in growth experiments and both species were resistant towards 0.05% CA. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis (424 down-expressed proteins with DCA, 376 down-expressed proteins with CDCA) and in E. faecium (362 down-expressed proteins with DCA, 391 down-expressed proteins with CDCA). Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a “general bile acid response”. Among these, various subunits of a V-type ATPase, different ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions instead of aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously.
Conclusions
Overall, these findings provide a detailed insight into the proteomic stress response of two Enterococcus species and help to understand the resistance potential and the stress-coping mechanisms of these important gastrointestinal bacteria.
... We wanted to evaluate if the inhibition of the V-ATPase pump could revert the TMZ resistance in the GSC lines. In this regard, we selected bafilomycin A1 (Baf) as an inhibitor of V-ATPase since it is the first-line molecule identified with this purpose and is efficient in a wide range of organisms at nanomolar concentrations [32]. Firstly, dose-response curves of nanomolar doses of Baf were tested for 48 h in order to identify a GSC line-specific sub-lethal dosage potentially effective in reverting TMZ resistance ( Figure 4). ...
The vacuolar proton-translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism underlying carcinogenesis is mainly based on anaerobic glycolytic reactions. Moreover, neoplastic cells use the V-ATPase to extrude chemotherapy drugs into the extra-cellular compartment as a drug resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1-induced inhibition of V-ATPase in patient-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 led to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models which are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem components, encouraging the search for novel strategies to limit its activity in order to circumvent resistance to conventional therapy.
... We wanted to evaluate if inhibition of V-ATPase pump could revert the TMZ resistance in GSC lines. In this regard, we selected bafilomycin A1 (Baf) as an inhibitor of V-ATPase since it is the firstline molecule identified with this purpose and it is efficient in a wide range of organisms at nanomolar concentrations [32]. Firstly, dose-response curves of nanomolar doses of Baf were tested for 48 h, in order to identify GSC line-specific sub-lethal dosage potentially effective in reverting the TMZ resistance ( Figure 4). ...
The vacuolar proton translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism is mainly based on anaerobic glycolytic reactions. Moreover, the neoplastic cells use the V-ATPase to extrude chemotherapy drugs to the extra-cellular compartment, as a drug-resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1 induced-inhibition of V-ATPase in patients-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome the resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 lead to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models that are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem component, encouraging the search for novel strategies to limit its activity in order to get around the resistance to the conventional therapy.
... To investigate whether failure to detect intracellular PrP d with N-terminal mAbs is due to proteolytic processing, we tested whether an increase in the endosomal/lysosomal pH may restore intracellular PrP d labelling by 5B2. Activation of endosomal/lysosomal proteinases is tightly regulated by the intraorganellar pH 26 and bafilomycin A1 (BafA1), an inhibitor of V-ATPases prevents luminal acidification 27 . Notably, dissipation of the luminal pH with BafA1 and the lysosomotropic agent NH 4 Cl gave rise to intracellular 5B2-positive aggregates in prion-infected cells (Fig. 2F), suggesting that truncation of full-length (FL)-PrP d is blocked upon inhibition of pH-dependent proteinases, thus excluding crypticity as cause for the failure to detect intracellular FL-PrP d . ...
The self-templating nature of prions plays a central role in prion pathogenesis and is associated with infectivity and transmissibility. Since propagation of proteopathic seeds has now been acknowledged a principal pathogenic process in many types of dementia, more insight into the molecular mechanism of prion replication is vital to delineate specific and common disease pathways. By employing highly discriminatory anti-PrP antibodies and conversion-tolerant PrP chimera, we here report that de novo PrP conversion and formation of fibril-like PrP aggregates are distinct in mechanistic and kinetic terms. De novo PrP conversion occurs within minutes after infection at two subcellular locations, while fibril-like PrP aggregates are formed exclusively at the plasma membrane, hours after infection. Phenotypically distinct pools of abnormal PrP at perinuclear sites and the plasma membrane show differences in N-terminal processing, aggregation state and fibril formation and are linked by exocytic transport via synaptic and large-dense core vesicles.
... In contrast, CB6 treatment of cells expressing the R685A spike mutant showed no cleavage of full-length spike in the cell lysates, nor culture supernatants; yet we detected noticeable amount of CB6 IgG heavy chain from the cell lysates (Fig 3A, bottom), suggested that binding of CB6 onto spike without the furin-cleaved S1/S2 site led to antibody internalization into the cells; both WT and R685A spike exhibited the cleavage of S2' similar to the Fig 2G. Moreover, when organelle acidification was inhibited by a vacuolar-ATPase inhibitor-bafilomycin A1 (BafA1) [45], we detected or presence of 10 μg/mL proteinase K for 30 min at 37˚C. blots are representative of two individual repeats; (C) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, IgG Hc, full-length spike, S1, S2 and cleaved S2' collected from supernatant and cell lysate fractions of CB6-stimulated HEK293T cells. ...
The COVID pandemic fueled by emerging SARS-CoV-2 new variants of concern remains a major global health concern, and the constantly emerging mutations present challenges to current therapeutics. The spike glycoprotein is not only essential for the initial viral entry, but is also responsible for the transmission of SARS-CoV-2 components via syncytia formation. Spike-mediated cell-cell transmission is strongly resistant to extracellular therapeutic and convalescent antibodies via an unknown mechanism. Here, we describe the antibody-mediated spike activation and syncytia formation on cells displaying the viral spike. We found that soluble antibodies against receptor binding motif (RBM) are capable of inducing the proteolytic processing of spike at both the S1/S2 and S2’ cleavage sites, hence triggering ACE2-independent cell-cell fusion. Mechanistically, antibody-induced cell-cell fusion requires the shedding of S1 and exposure of the fusion peptide at the cell surface. By inhibiting S1/S2 proteolysis, we demonstrated that cell-cell fusion mediated by spike can be re-sensitized towards antibody neutralization in vitro . Lastly, we showed that cytopathic effect mediated by authentic SARS-CoV-2 infection remain unaffected by the addition of extracellular neutralization antibodies. Hence, these results unveil a novel mode of antibody evasion and provide insights for antibody selection and drug design strategies targeting the SARS-CoV-2 infected cells.
... To establish a direct association with V-ATPase, we treated wild-type with a specific V-ATPase inhibitor, bafilomycin A1. The concentration of bafilomycin A1 utilized in this study was 500 nM, which has been previously reported to specifically inhibit V-ATPase at this dose [41,42]. By treating the wild-type strain with this inhibitor, we aimed to determine if it could lead to rav1Δ-like phenotypic changes in wild-type C. neoformans. ...
V-ATPase, which comprises 13–14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE ( r egulator of H ⁺ - A TPase of v acuolar and e ndosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans , and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C . neoformans . The hRAVE complex played a critical role in the pathogenicity of C . neoformans , and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis.
... This response was suppressed by the knockdown of ATP6V0C and ATP6V0D1 (Fig. 1a and Supplementary Fig. S1a), two core subunits of the v-ATPase complex 26,33 . Similarly, inhibition of v-ATPase activity by two small-molecule inhibitors, Bafilomycin A1 (BafA1) and Concanamycin A (ConA) 34,35 , strongly attenuated Dox-induced expression of typical UPR mt genes in mouse embryonic fibroblasts (MEFs) (Fig. 1b and Supplementary Fig. S1b). Among these approaches to suppress UPR mt activation, ConA treatment in MEFs was the most efficacious (Fig. 1b). ...
Lysosomes are central platforms for not only the degradation of macromolecules but also the integration of multiple signaling pathways. However, whether and how lysosomes mediate the mitochondrial stress response (MSR) remain largely unknown. Here, we demonstrate that lysosomal acidification via the vacuolar H ⁺ -ATPase (v-ATPase) is essential for the transcriptional activation of the mitochondrial unfolded protein response (UPR mt ). Mitochondrial stress stimulates v-ATPase-mediated lysosomal activation of the mechanistic target of rapamycin complex 1 (mTORC1), which then directly phosphorylates the MSR transcription factor, activating transcription factor 4 (ATF4). Disruption of mTORC1-dependent ATF4 phosphorylation blocks the UPR mt , but not other similar stress responses, such as the UPR ER . Finally, ATF4 phosphorylation downstream of the v-ATPase/mTORC1 signaling is indispensable for sustaining mitochondrial redox homeostasis and protecting cells from ROS-associated cell death upon mitochondrial stress. Thus, v-ATPase/mTORC1-mediated ATF4 phosphorylation via lysosomes links mitochondrial stress to UPR mt activation and mitochondrial function resilience.
... Nutrient deprivation in combination with Bafilomycin A1 (BafA) treatment is a widely used method for investigating autophagy 37 . While the starvation induces metabolic changes including autophagy 38 , the BafA treatment increases endo-lysosomal pH by inhibiting vATPase function and thus prevents lysosomal protein degradation 39,40 . This helps to gauge autophagic flux and facilitates the capture of autophagic structures by imaging 37 . ...
The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.
... The incubation medium contained cell samples, 5 mM ATP, 5 mM MgSO 4 , and buffer solutions according to pH optima for three major yeast ATPases: 20 mM MES-NaOH buffer, pH 6.5 for the cell membrane ATPase; 50 mM Tris-HCl, pH 7.0 for the vacuolar membrane ATPase; and 50 mM Tris-HCl, pH 8.5 for the mitochondrial ATPase. Activity of these ATPases was determined using specific inhibitors: 100 μM Na 3 VO 4 ·12H 2 O as an inhibitor of the cell membrane ATPase (Borst-Pauwels, Peters, 1981); 0.5 μM bafilomycin A (Sigma-Aldrich, United States) as an inhibitor of the vacuolar membrane ATPase (Bowman et al., 1988); and 5 mM NaN 3 as an inhibitor of the mitochondrial ATPase (Harris, 1989). In all three cases, ATP hydrolysis was carried out at 30°C. ...
The Ррх1 exopolyphosphatase of yeast is a constitutive protein localized predominantly in the
cytoplasm. The purified enzyme hydrolyzes inorganic polyphosphates with high activity; however, in the
knockout Δppx1 mutant of Saccharomyces cerevisiae the increase in the polyphosphate level was small, and
no changes in physiological properties of this mutant were observed. To elucidate the functions of Ppx1, we
studied the physiological characteristics of the S. cerevisiae strain overexpressing this enzyme. When cultivated
in the YPD medium, the strain overexpressing Ppx1 showed no growth features different from those of
the parental strain. The following physiological features of the strain overexpressing Ppx1 were observed at
the stationary stage of growth: the level of ATP increased by nine times, the activity of vacuolar ATPase significantly
decreased, and the sensitivity to peroxide increased compared to the parental strain. The level of
reactive oxygen species doubled, while the degree of lipid oxidation remained the same as in parental strain.
Since overexpression of Ppx1 under the culture conditions used did not affect the polyphosphate level, these
polymers were not the regulators of the changes described above. Response to oxidative stress and vacuolar
ATPase activity in yeasts is known to be regulated by cAMP, while Ppx1 is capable of hydrolyzing this signaling
compound. We suggest that one of the functions of Ppx1 in yeasts is participation in the regulation of
cAMP level.
... Neither Kifunensine, an inhibitor of N-glycosylation, nor Brefeldin A, a disruptor of Golgi structure and function, decreased lysosome spot intensity relative to the negative control ( Figure 5B) [13,14]. Bafilomycin A1, an inhibitor of V-ATPases found on endosomes, potently decreases lysotracker spot intensity, indicating increased endosome pH ( Figure 5B) [15]. Although ZJ-101 does not affect lysosome staining intensity, the average lysosome puncta area is significantly (p = 0.02) decreased ( Figure 5C) and is shared by brefeldin A and kifunensine treatments. ...
Marine natural products represent a unique source for clinically relevant drugs due to their vast molecular and mechanistic diversity. ZJ-101 is a structurally simplified analog of the marine natural product superstolide A, isolated from the New Caledonian sea sponge Neosiphonia Superstes. The mechanistic activity of the superstolides has until recently remained a mystery. Here, we have identified potent antiproliferative and antiadhesive effects of ZJ-101 on cancer cell lines. Furthermore, through dose–response transcriptomics, we found unique dysregulation of the endomembrane system by ZJ-101 including a selective inhibition of O-glycosylation via lectin and glycomics analysis. We applied this mechanism to a triple-negative breast cancer spheroid model and identified a potential for the reversal of 3D-induced chemoresistance, suggesting a potential for ZJ-101 as a synergistic therapeutic agent.
... The structure of 6 represents a 16-membered macrolide with a tetraene core, a cyclic hemiacetal, a C14 methoxy, and a C23-isopropyl group within its polypropionate chain. Bafilomycin A 1 6 exhibited potent antifungal and antibacterial activity and is also the first known inhibitor of Vacuolar H + -ATPase (VATPase) [47]. Protomycinolide IV (7) (Figure 4) is the aglycone of Mycinamicins [48]. ...
Polypropionate units are a common structural feature of many of the natural products in polyketides, some of which have shown a broad range of antimicrobial and therapeutic potential. Polypropionates are composed of a carbon skeleton with alternating methyl and hydroxy groups with a specific configuration. Different approaches have been developed for the synthesis of polypropionates and herein we include, for the first time, all of the epoxide-based methodologies that have been reported over the years by several research groups such as Kishi, Katsuki, Marashall, Miyashita, Prieto, Sarabia, Jung, McDonald, etc. Several syntheses of polypropionate fragments and natural products that employed epoxides as key intermediates have been described and summarized in this review. These synthetic approaches involve enatio- and diastereoselective synthesis of epoxides (epoxy-alcohols, epoxy-amides, and epoxy-esters) and their regioselective cleavage with carbon and/or hydride nucleophiles. In addition, we included a description of the isolation and biological activities of the polypropionates and related natural products that have been synthetized using epoxide-based approaches. In conclusion, the epoxide-based methodologies are a non-aldol alternative approach for the construction of polypropionate.
... 16 Similar pegs have been reported in Naegleria as well, 16 consistent with our identification of a full complement of vacuolar-type H+ATPase subunits along with aquaporins in the Naegleria genome ( Table 1, Fig. S1B-C). To directly test if these proton pumps are required for contractile vacuole function in Naegleria, we treated cells with the vacuolar-type H+ATPase inhibitor Bafilomycin A1. 62,63 Treatment with 100 nM Bafilomycin A1 prevented the refilling of contractile vacuoles (Fig. 1D, Fig. S1D), consistent with vacuolar-type proton pump activity facilitating water flow from the cytosol into the contractile vacuole. ...
Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like freshwater must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify basic principles governing contractile vacuole function, we here investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages—the discoban Naegleria gruberi , and the amoebozoan slime mold Dictyostelium discoideum . Using quantitative cell biology we find that, although these species respond differently to osmotic challenges, they both use actin for osmoregulation, as well as vacuolar-type proton pumps for filling contractile vacuoles. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum . Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
... The macrolide antibiotic bafilomycin A1 acts as a specific inhibitor of the lysosomal vacuolar-type H + -ATPase (V-ATPase), which is required for the acidification of endo-lysosomal lumen (Bowman et al 1988, Dröse and Altendorf 1997, Huss et al 2011). Bafilomycin A1 inhibits V-ATPases with a high affinity, at nanomolar concentrations (Bowman et al 1988). It has been suggested that the pharmacological inhibition of H + uptake into the lumen via the V-ATPase leads to passive loss of H + through leaks, which in turn dissipates the H + gradient across the lysosomal membrane, leading to luminal alkalization. ...
Autophagy, vital for removing cellular waste, is triggered differently by small molecules and nanoparticles. Small molecules, like rapamycin, non‐selectively activate autophagy by inhibiting the mTOR pathway, which is essential for cell regulation. This can clear damaged components but may cause cytotoxicity with prolonged use. Nanoparticles, however, induce autophagy, often causing oxidative stress, through broader cellular interactions and can lead to a targeted form known as “xenophagy.” Their impact varies with their properties but can be harnessed therapeutically. In this review, the autophagy induced by nanoparticles is explored and small molecules across four dimensions: the mechanisms behind autophagy induction, the outcomes of such induction, the toxicological effects on cellular autophagy, and the therapeutic potential of employing autophagy triggered by nanoparticles or small molecules. Although small molecules and nanoparticles each induce autophagy through different pathways and lead to diverse effects, both represent invaluable tools in cell biology, nanomedicine, and drug discovery, offering unique insights and therapeutic opportunities.
New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite’s plasma membrane Na⁺-extruding, H⁺-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite’s internal [Na⁺] and pH. Here, we designed a “pH fingerprint” assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H⁺ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H⁺ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H⁺ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl– transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.
MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.
The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene KO (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia, which was produced from l-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome–lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.
Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal‐lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP‐3 complex mu subunit, the lysosomal‐trafficking regulator LYST, mucopilin‐1, and the Wiskott–Aldrich syndrome‐associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation‐independent mannose‐6‐phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.
The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These ‘HOPS bodies’ were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.
Free polymannose-type oligosaccharides (fOS) are processed by cytosolic enzymes to generate Man5GlcNAc which is transferred to lysosomes and degraded. Lysosomal fOS import was demonstrated in vitro but is poorly characterized in part due to lack of convenient substrates. As chitooligosaccharides (COS, oligomers β1,4-linked GlcNAc) block [3H]Man5GlcNAc transport into lysosomes, we asked if COS are themselves transported and if so, can they be chemically modified to generate fluorescent substrates.
We show that COS are degraded by lysosomal hydrolases to generate GlcNAc, and robust ATP-dependent transport of [3H]COS2/4 di and tetrasaccharides into intact rat liver lysosomes was observed only after blocking lysosomal [3H]GlcNAc efflux with cytochalasin B. As oligosaccharides with unmodified reducing termini are the most efficient inhibitors of [3H]COS2/4 and [3H]Man5GlcNAc transport, the non-reducing GlcNAc residue of COS2-4 was de-N-acetylated using Sinorhizobium meliloti NodB, and the resulting amine substituted with rhodamine B (RB) to yield RB-COS2-4. The fluorescent compounds inhibit [3H]Man5GlcNAc transport and display temperature-sensitive, ATP-dependent transport into a sedimentable compartment that is ruptured with the lysosomotropic agent L-methyl methionine ester. Once in this compartment, RB-COS3 is converted to RB-COS2 further identifying it as the lysosomal compartment. RB-COS2/3 and [3H]Man5GlcNAc transports are blocked similarly by competing sugars, and are partially inhibited by the vacuolar ATPase inhibitor bafilomycin and high concentrations of the P-type ATPase inhibitor orthovanadate.
These data show that Man5GlcNAc, COS2/4 and RB-COS2/3 are transported into lysosomes by the same or closely related mechanism and demonstrate the utility of COS modified at their non-reducing terminus to study lysosomal oligosaccharide transport.
Phosphorodiamidate morpholino oligomers (PMOs) are a special type of antisense oligonucleotides (ASOs) that can be used as therapeutic modulators of pre-mRNA splicing. Application of nucleic-acid-based therapeutics generally requires suitable delivery systems to enable efficient transport to intended tissues and intracellular targets. To identify potent formulations of PMOs, we established a new in vitro–in vivo screening platform based on mdx exon 23 skipping. Here, a new in vitro positive read-out system (mCherry-DMDEx23) is presented that is sensitive toward the PMO(Ex23) sequence mediating DMD exon 23 skipping and, in this model, functional mCherry expression. After establishment of the reporter system in HeLa cells, a set of amphiphilic, ionizable xenopeptides (XPs) was screened in order to identify potent carriers for PMO delivery. The identified best-performing PMO formulation with high splice-switching activity at nanomolar concentrations in vitro was then translated to in vivo trials, where exon 23 skipping in different organs of healthy BALB/c mice was confirmed. The predesigned in vitro–in vivo workflow enables evaluation of PMO(Ex23) carriers without change of the PMO sequence and formulation composition. Furthermore, the identified PMO–XP conjugate formulation was found to induce highly potent exon skipping in vitro and redistributed PMO activity in different organs in vivo.
Autophagy is an efficient and attractive protein degradation pathway in addition to the ubiquitin-proteasome system. Herein, systematic optimization of coumarin analogs linked with the CDK9 inhibitor SNS-032 is reported that may bind to cyclin-dependent kinase 9 (CDK9) and microtubule-associated protein 1 light chain 3 beta (LC3B) simultaneously, which leads to the selective autophagic degradation of targeted CDK9/cyclin T1 and is different from the PROTAC degrader THAL-SNS-032. Further mechanism studies revealed an autophagy-lysosome pathway, where the degraders possibly formed a ternary complex with CDK9 and LC3B. In addition, degrader 10 showed antitumor efficacy in vivo. Our work optimized a potent LC3B recruiter and demonstrated the feasibility of autophagy-tethering compounds (ATTECs), which could be applied for the degradation of diverse intracellular pathogenic proteins to treat related diseases.
The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a pro-survival program and to sustain a pro-angiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we discovered that decorin downregulated a cluster of tumor-associated genes involved in lymphatic vessel development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of lymphatic vessels, were markedly suppressed at both the mRNA and protein levels and this suppression correlated with a significant reduction in tumor lymphatic vessels. We further discovered that soluble decorin, but not its homologous proteoglycan biglycan, inhibited lymphatic vessel sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with VEGFR3, the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we discovered that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a new biological factor with anti-lymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.
Identifying the mechanisms by which bacterial pathogens kill host cells is fundamental to understanding how to control and prevent human and animal disease. In the case of Bacillus thuringiensis (Bt), such knowledge is critical to using the bacterium to kill insect vectors that transmit human and animal disease. For the Cry4B toxin produced by Bt, its capacity to kill Anopheles gambiae, the primary mosquito vector of malaria, is the consequence of a variety of signaling activities. We show here that Cry4B, acting as first messenger, binds specifically to the bitopic cadherin BT-R3 G-protein-coupled receptor (GPCR) localized in the midgut of A. gambiae, activating the downstream second messenger cyclic adenosine monophosphate (cAMP). The direct result of the Cry4B-BT-R3 binding is the release of αs from the heterotrimeric αβγ-G-protein complex and its activation of adenylyl cyclase (AC). The upshot is an increased level of cAMP, which activates protein kinase A (PKA). The functional impact of cAMP-PKA signaling is the stimulation of Na+/K+-ATPase (NKA) which serves as an Na+/K+ pump to maintain proper gradients of extracellular Na+ and intracellular K+. Increased level of cAMP amplifies NKA and upsets normal ion concentration gradients. NKA, as a scaffolding protein, accelerates the first messenger signal to the nucleus, generating additional BT-R3 molecules and promoting their exocytotic trafficking to the cell membrane. Accumulation of BT-R3 on the cell surface facilitates recruitment of additional toxin molecules which, in turn, amplify the original signal in a cascade-like manner. This report provides the first evidence of a bacterial toxin using NKA via AC/PKA signaling to execute cell death.
Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30 - 50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.
Nocardia are opportunistic human pathogens that can cause a range of debilitating and difficult to treat infections of the lungs, brain, skin, and soft tissues. Despite their close relationship to the well-known secondary metabolite-producing genus, Streptomyces, comparatively few natural products are known from the Nocardia, and even less is known about their involvement in the pathogenesis. Here, we combine chemistry, genomics, and molecular microbiology to reveal the production of terpenomycin, a new cytotoxic and antifungal polyene from a human pathogenic Nocardia terpenica isolate. We unveil the polyketide synthase (PKS) responsible for terpenomycin biosynthesis and show that it combines several unusual features, including "split", skipped, and iteratively used modules, and the use of the unusual extender unit methoxymalonate as a starter unit. To link genes to molecules, we constructed a transposon mutant library in N. terpenica, identifying a terpenomycin-null mutant with an inactivated terpenomycin PKS. Our findings show that the neglected actinomycetes have an unappreciated capacity for the production of bioactive molecules with unique biosynthetic pathways waiting to be uncovered and highlights these organisms as producers of diverse natural products.
Immune responses highly depend on the effective trafficking of immune cells into and within secondary lymphoid organs (SLOs). Atypical chemokine receptors (ACKRs) scavenge chemokines to eliminate them from the extracellular space, thereby generating gradients that guide leukocytes. In contrast to canonical chemokine receptors, ACKRs do not induce classical intracellular signaling that results in cell migration. Recently, the closest relative of ACKR3, GPR182, has been partially deorphanized as a potential novel ACKR. We confirm and extend previous studies by identifying further ligands that classify GPR182 as a broadly scavenging chemokine receptor. We validate the “atypical” nature of the receptor, wherein canonical G-protein-dependent intracellular signaling is not activated following ligand stimulation. However, β-arrestins are required for ligand-independent internalization and chemokine scavenging whereas the C-terminus is in part dispensable. In the absence of GPR182 in vivo, we observed elevated chemokine levels in the serum but also in SLO interstitium. We also reveal that CXCL13 and CCL28, which do not bind any other ACKR, are bound and efficiently scavenged by GPR182. Moreover, we found a cooperative relationship between GPR182 and ACKR3 in regulating serum CXCL12 levels, and between GPR182 and ACKR4 in controlling CCL20 levels. Furthermore, we unveil a new phenotype in GPR182-KO mice, in which we observed a reduced marginal zone (MZ), both in size and in cellularity, and thus in the T-independent antibody response. Taken together, we and others have unveiled a novel, broadly scavenging chemokine receptor, which we propose should be named ACKR5.
Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
Nucleic acid-based medicines and vaccines are becoming an important part of our therapeutic toolbox. One key genetic medicine is antisense oligonucleotides (ASOs), which are short single-stranded nucleic acids that downregulate protein production by binding to mRNA. However, ASOs cannot enter the cell without a delivery vehicle. Diblock polymers containing cationic and hydrophobic blocks self-assemble into micelles that have shown improved delivery compared to linear nonmicelle variants. Yet synthetic and characterization bottlenecks have hindered rapid screening and optimization. In this study, we aim to develop a method to increase throughput and discovery of new micelle systems by mixing diblock polymers together to rapidly form new micelle formulations. We synthesized diblocks containing an n-butyl acrylate block chain extended with cationic moieties amino ethyl acrylamide (A), dimethyl amino ethyl acrylamide (D), or morpholino ethyl acrylamide (M). These diblocks were then self-assembled into homomicelles (A100, D100, and M100)), mixed micelles comprising 2 homomicelles (MixR%+R'%), and blended diblock micelles comprising 2 diblocks blended into one micelle (BldR%R'%) and tested for ASO delivery. Interestingly, we observed that mixing or blending M with A (BldA50M50 and MixA50+M50) did not improve transfection efficiency compared to A100; however, when M was mixed with D, there was a significant increase in transfection efficacy for the mixed micelle MixD50+M50 compared to D100. We further examined mixed and blended D systems at different ratios. We observed a large increase in transfection and minimal change in toxicity when M was mixed with D at a low percentage of D incorporation in mixed diblock micelles (i.e., BldD20M80) compared to D100 and MixD20+M80. To understand the cellular mechanisms that may result in these differences, we added proton pump inhibitor Bafilomycin-A1 (Baf-A1) to the transfection experiments. Formulations that contain D decreased in performance in the presence of Baf-A1, indicating that micelles with D rely on the proton sponge effect for endosomal escape more than micelles with A. This result supports our conclusion that M is able to modulate transfection of D, but not with A. This research shows that polymer blending in a manner similar to that of lipids can significantly boost transfection efficiency and is a facile way to increase throughput of testing, optimization, and successful formulation identification for polymeric nucleic acid delivery systems.
It had been shown that lentinan (LNT) was mainly distributed in the liver after intravenous administration. The study aimed to investigate the integrated metabolic processes and mechanisms of LNT in the liver, as these have not been thoroughly explored. In current work, 5-([4,6-dichlorotriazin-2-yl] amino) fluorescein and cyanine 7 were used to label LNT for tracking its metabolic behavior and mechanisms. Near-infrared imaging demonstrated that LNT was captured mainly by the liver. Kupffer cell (KC) depletion reduced LNT liver localization and degradation in BALB/c mice. Moreover, experiments with Dectin-1 siRNA and Dectin-1/Syk signaling pathway inhibitors showed that LNT was mainly taken up by KCs via the Dectin-1/Syk pathway and promoted lysosomal maturation in KCs via this same pathway, which in turn promoted LNT degradation. These empirical findings offer novel insights into the metabolism of LNT in vivo and in vitro, which will facilitate the further application of LNT and other β-glucans.
Triple-negative breast cancer (TNBC) is a leading malignancy among women that currently lack effective targeted therapeutic agents, and the limitations of treatment have prompted the emergence of new strategies. Methuosis is a novel vacuole-presenting cell death modality that promotes tumor cell death. Hence, a series of pyrimidinediamine derivatives were designed and synthesized through evaluation of their abilities that inhibit proliferation as well as induce methuosis against TNBC cells. Among them, JH530 showed excellent anti-proliferative activities and vacuolization capacity in TNBC. The mechanism research indicated that JH530 caused cell death through inducing methuosis of cancer cells. Furthermore, JH530 inhibited tumor growth remarkably in the HCC1806 xenograft model without an apparent decrease in body weight. Overall, JH530 is a methuosis inducer that displayed remarkable suppression of TNBC growth in vitro and in vivo, which provides a basis for the future progress of more small molecules for TNBC treatment.
Mutations in the human ATP13A2 (PARK9), a lysosomal ATPase, cause Kufor-Rakeb Syndrome, an early-onset form of Parkinson’s disease (PD). Here, we demonstrate that ATP13A2 functions as a lysosomal H⁺,K⁺-ATPase. The K⁺-dependent ATPase activity and the lysosomal K⁺-transport activity of ATP13A2 are inhibited by an inhibitor of sarco/endoplasmic reticulum Ca²⁺-ATPase, thapsigargin, and K⁺-competitive inhibitors of gastric H⁺,K⁺-ATPase, such as vonoprazan and SCH28080. Interestingly, these H⁺,K⁺-ATPase inhibitors cause lysosomal alkalinization and α-synuclein accumulation, which are pathological hallmarks of PD. Furthermore, PD-associated mutants of ATP13A2 show abnormal expression and function. Our results suggest that the H⁺/K⁺-transporting function of ATP13A2 contributes to acidification and α-synuclein degradation in lysosomes.
Simple Summary
A selected set of natural products that target the structure and function of the Golgi apparatus in cancer cells is highlighted in this review. The potential for modulating these Golgi-mediated signaling pathways for anticancer drug development is also discussed.
Abstract
The Golgi apparatus plays an important role in maintaining cell homeostasis by serving as a biosynthetic center for glycans, lipids and post-translationally modified proteins and as a sorting center for vesicular transport of proteins to specific destinations. Moreover, it provides a signaling hub that facilitates not only membrane trafficking processes but also cellular response pathways to various types of stresses. Altered signaling at the Golgi apparatus has emerged as a key regulator of tumor growth and survival. Among the small molecules that can specifically perturb or modulate Golgi proteins and organization, natural products with anticancer property have been identified as powerful chemical probes in deciphering Golgi-related pathways and, in particular, recently described Golgi stress response pathways. In this review, we highlight a set of Golgi-targeting natural products that enabled the characterization of the Golgi-mediated signaling events leading to cancer cell death and discuss the potential for selectively exploiting these pathways for the development of novel chemotherapeutic agents.
Hexacosalactone A (1) is a polyene macrolide compound featuring a 2-amino-3-hydroxycyclopent-2-enone (C5N)-fumaryl moiety. While compound 1 has been proposed to be assembled via a type I modular polyketide synthase (PKS) system, most of the putative biosynthetic steps lack experimental evidence. In this study, we elucidated the post-PKS tailoring steps of compound 1 through in vivo gene inactivation and in vitro biochemical assays. We demonstrated that the amide synthetase HexB and O-methyltransferase HexF are responsible for the installations of the C5N moiety and the methyl group at 15-OH of compound 1, respectively; two new hexacosalactone analogs, named hexacosalactones B (4) and C (5), were purified and structurally characterized, followed by anti-multidrug resistance (anti-MDR) bacterial assays, revealing that the C5N ring and the methyl group are necessary for the antibacterial bioactivities. Through database mining of C5N-forming proteins HexABC, six uncharacterized biosynthetic gene clusters (BGCs), putatively encoding compounds with different types of backbones, were identified, providing potentials to discover novel bioactive compounds with C5N moiety.
IMPORTANCE In this study, we elucidate the post-PKS tailoring steps during the biosynthesis of compound 1 and demonstrate that both C5N and 15-OMe groups are critical for the antibacterial activities of compound 1, paving the way for generation of hexacosalactone derivatives via synthetic biology strategy. In addition, mining of HexABC homologs from the GenBank database revealed their wide distribution across the bacterial world, facilitating the discovery of other bioactive natural products with C5N moiety.
Mutants of Neurospora crassa have been isolated that are highly resistant to inhibition by oligomycin, an inhibitor of mitochondrial ATPase activity. Dixon plots (Dixon, M., and Webb, E.C. (1964) Enzymes, 2nd Ed, pp. 328-330, Academic Press, New York) of oligomycin inhibition curves of the parent strain and the resistant mutants are linear, indicating that oligomycin interacts at a single site within the ATPase complex. The Ki values obtained from the mutants vary from 150 to 900 times greater than the Ki obtained for the parent strain. The parent strain and the oligomycin-resistant mutants are also inhibited by bathophenanthroline, a lipophilic chelating agent that inhibits F1 ATPase activity. Dixon plots of bathophenanthroline inhibition curves are also linear and Ki values obtained are all approximately equal. Crosses of the oligomycin-resistant mutants to the oligomycin-sensitive parent strain show a mendelian segregation of the resistance characteristic. These data show that mutations leading to oligomycin resistance in Neurospora are due to alterations in nuclear genes.
Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.
The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.
Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.
The proton-ATPase of chromaffin granules was purified so as to maintain its proton-pumping activity when reconstituted into phospholipid vesicles. The purification procedure involved solubilization with polyoxyethylene 9 lauryl ether, hydroxylapatite column, precipitation by ammonium sulfate, and glycerol gradient centrifugation. The protease inhibitor mixture used in previous studies inhibited the proton-pumping activity of the enzyme; therefore, the protein was stabilized by pepstatin A and leupeptin. The enzyme was purified at least 50-fold with respect to both ATPase and proton-pumping activity. The ATP-dependent proton uptake activity of the reconstituted enzyme was absolutely dependent on the presence of Cl- or Br- outside the vesicles, whereas sulfate, acetate, formate, nitrate, and thiocyanate were inhibitory. Sulfate inhibition seems to be due to competition with Cl- on the anion-binding site outside the vesicles, whereas nitrate and thiocyanate inhibited only from the internal side. As with the inhibition by N-ethylmaleimide, the proton-pumping activity was much more sensitive to nitrate than the ATPase activity. About 20 mM nitrate were sufficient for 90% inhibition of the proton-pumping activity while 100 mM inhibited only 50% of the ATPase activity both in situ and in the reconstituted enzyme. The possible regulatory effect of anions on the ATP-dependent proton uptake in secretory granules is discussed.
2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F1 ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the beta subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed beta-subunit Tyr-368 to be present at noncatalytic sites and beta Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.
The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.
Vacuolar ATPases constitute a novel class of N-ethylmaleimide- and nitrate-sensitive proton pumps associated with the endomembrane system of eukaryotic cells. They resemble F0F1-ATPases in that they are large multimeric proteins, 400-500 kDa, composed of three to nine different subunits. Previous studies have indicated that the active site is located on the approximately 70-kDa subunit. Using antibodies to the approximately 70-kDa subunit of corn to screen a carrot root lambda gt11 cDNA library, we have isolated cDNA clones of the carrot 69-kDa subunit. The complete primary structure of the 69-kDa subunit was then determined from the nucleotide sequence of its cDNA. The 69-kDa subunit consists of 623 amino acids (Mr 68,835), with no obvious membrane-spanning regions. The carrot cDNA sequence was over 70% homologous with exons of a Neurospora 69-kDa genomic clone. The protein sequence of the carrot 69-kDa subunit also exhibited 34.3% identity to four representative F0F1-ATPase beta-chains over a 275-amino-acid core stretch of similar sequence. Alignment studies revealed several regions which were highly homologous to beta-chains, including sequences previously implicated in catalytic function. This provides definitive evidence that the vacuolar ATPase is closely related to the F0F1-type ATPases. A major functional difference between the 69-kDa and beta-subunits is the location of 3 critical cysteine residues: two in the putative catalytic region (Cys-248 and Cys-256) and one in the proposed Mg2+-binding site (Cys-279). These cysteines (and two others) probably account for the sensitivity of the vacuolar H+-ATPase to the sulfhydryl reagent, N-ethylmaleimide. It is proposed that the two ATPases may have arisen from a common ancestor by the insertion or deletion of a large stretch of nonhomologous sequence near the amino-terminal end of the subunit.
The vanadate-sensitive ATPase of Streptococcus faecalis, purified to homogeneity, was reconstituted into soybean phospholipid vesicles in a functional state. Freeze-fracture electron micrographs revealed a relatively uniform population of unilamellar liposomes of 50-100 nm in diameter, with particles protruding from both fracture faces. Transport studies with 42K+ and with a K+-selective electrode showed that the ATP-ase catalyzes electrogenic potassium extrusion in proteoliposomes. The following parameters for potassium transport in the reconstituted system were determined: K+/ATP stoichiometry = 1, Km for potassium = 1.4 mM, Vmax = 0.1 mumol/min/mg. The ATPase could be activated by an electrical membrane potential, vesicle interior positive. This ATPase thus appears to function as a potential regulated, ATP-driven pump that serves in electrogenic potassium accumulation by the bacterial cell.
We isolated a large number of mutations in the structural gene for the plasma membrane ATPase (PMA1) of Saccharomyces cerevisiae.
These mutations were selected by their resistance to the aminoglycoside antibiotic hygromycin B. Biochemical analysis of purified
membrane preparations showed that the plasma membrane ATPase activity of the mutants was reduced as much as 75%. Intragenic
complementation of pma1 mutants suggested that the yeast plasma membrane ATPase was a multimeric enzyme. The pma1 mutants
were apparently defective in maintaining internal pH; more than half of the mutants were unable to grow either at a low pH
or in the presence of a weak acid. Most pma1 mutants were also osmotic pressure sensitive. At a very low temperature (5 degrees
C) many pma1 mutants were unable to grow and were arrested as unbudded cells. The three most severely affected mutants were
also unable to grow in the presence of NH4+. The most extreme mutant exhibited a severe defect in progression through the
cell cycle; on synthetic medium, the cells progressively accumulated nucleus-containing small buds that generally failed to
complete bud enlargement and cytokinesis. Most of the pleiotropic phenotypes of pma1 mutants could be suppressed by the addition
of 50 mM KCl but not NaCl to the medium.
Chromaffin-granule membranes contain two ATPases, which can be separated by (NH4)2SO4 fractionation after solubilization with detergents, or by phase segregation in Triton X-114. ATPase I (Mr 400000) is inhibited by trialkyltin, quercetin and alkylating agents, and hydrolyses both ATP and ITP. It contains up to five types of subunit, including a low-Mr hydrophobic polypeptide that reacts with dicyclohexylcarbodi-imide; these subunits are unrelated to those of mitochondrial F1F0-ATPase, as judged by size and reaction with antibodies. ATPase II (Mr 140000) is inhibited by vanadate, and is specific for ATP; it has not been extensively purified. Proton translocation by resealed chromaffin-granule 'ghosts', measured by uptake of methylamine or by quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine, is supported by the hydrolysis of ATP or ITP, and inhibited by quercetin or alkylating agents, but not by vanadate. ATPase I must therefore be the proton translocator involved in the uptake of catecholamines and possibly of other components of the chromaffin-granule matrix, whereas ATPase II does not translocate protons.
The H+-translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of Mr approximately equal to 70,000 and approximately equal to 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[14C]ethylmaleimide and 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole, labeled the Mr approximately equal to 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[14C]dicyclohexylcarbodiimide labeled a polypeptide of Mr approximately equal to 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of Mr 5.2 X 10(5), 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochondrial ATPase but strongly crossreacted with antiserum against a polypeptide of Mr approximately equal to 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.
The vanadate-sensitive membrane ATPase of Streptococcus faecalis forms, as part of the reaction cycle, an acylphosphate intermediate. The phosphorylated amino acid residue was identified by reducing the purified reconstituted phosphoenzyme with [3H]borohydride, followed by acid hydrolysis of the protein and quantitative amino acid analysis. Tritiated homoserine was found to be the resulting reaction product, generated through the reduction of a beta-aspartyl phosphate residue. The S. faecalis ATPase thus forms the same phosphorylated intermediate as a number of eukaryotic transport ATPases and appears to be related to these enzymes.
We have purified a novel membrane ATPase from Streptococcus faecalis by the following procedure: extraction of membranes with Triton X-100 followed by fractionation of the extract by successive DEAE-cellulose chromatography, hydroxylapatite chromatography and Cm-Sepharose chromatography. The overall yield was 5%. The purified ATPase appears to consist of a single polypeptide component of Mr = 78,000. The Triton-solubilized purified enzyme has a specific activity of approximately 50 mumol of ATP hydrolyzed per min per mg, is dependent on phospholipids for activity, and is strongly inhibited by vanadate (I50 = 3 microM). Maximal ATPase activity is displayed at pH 7.3. Mg2+-ATP, for which the enzyme has a Km of 60 microM, is the best substrate. The ATPase forms an acylphosphate intermediate that can also be detected in native membranes as the major acylphosphate component. The purified ATPase, when reconstituted into soybean phospholipid vesicles, exhibits coupling, e.g. the ATPase activity can be stimulated at least 8-fold by valinomycin in the presence of potassium. Based on these observations we conclude that the enzyme we have purified is an ion-motive ATPase, most likely a K+-ATPase.
Pre-incubation of the plasma membrane [H+]-ATPase of Neurospora crassa with the sulfhydryl reagent, N-ethylmaleimide (NEM), leads to a marked inhibition of ATPase activity. Loss of activity depends upon preincubation pH and follows pseudo-first order kinetics with respect to NEM concentration. MgATP, the physiological substrate for ATPase activity, protects against NEM inactivation with an average dissociation constant of 1.5 mM at 0 degrees C. This value agrees well with the measured Km for MgATP hydrolysis (1.3 mM at 30 degrees C and 0.9 mM at 15 degrees C). MgADP also protects against NEM inhibition with an average KD of 0.18 mM at 0 degrees C; MgADP is a competitive inhibitor of enzyme activity, with a Ki of 0.08 mM at 30 degrees C and 0.09 mM at 15 degrees C. Free ATP and ADP, as well as other Mg nucleotides (MgGTP, MgCTP, and MgUTP) which are hydrolyzed at much slower rates than MgATP, exert smaller protective effects. These results suggest that nucleotides protect against NEM inhibition by binding to the catalytic site of the ATPase. NEM can therefore be used as a probe to study the nature of enzyme-ligand interactions.
The [H+]-ATPase of the Neurospora plasma membrane is composed of a single Mr = 104,000 polypeptide (B. J. Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. (1981) 256, 12343-12349). The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the ATPase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. The rate constant for inactivation at pH 7.5 and 30 degrees C is approximately 1000 M-1 min-1, similar to values reported for the DCCD-binding proteolipid of F0-F1-type [H+]-ATPases and for the sarcoplasmic reticulum [Ca+2]-ATPase. Although hydrophobic carbodiimides are inhibitory at micromolar concentrations, a hydrophilic analogue, 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, is completely inactive even at millimolar concentrations. This result implies that the DCCD-reactive site is located in a lipophilic environment. [14C]DCCD is incorporated into the Mr = 104,000 polypeptide at a rate similar to the rate of inactivation. There is no evidence for a separate low molecular weight DCCD-binding proteolipid. Using quantitative amino acid analysis, we established that complete inhibition occurs at a stoichiometry of 0.4 mol of DCCD/mol of polypeptide. Overall, the results are consistent with the idea that DCCD reacts with a single amino acid residue of the Neurospora [H+]-ATPase, thereby blocking ATP hydrolysis and proton translocation.
The plasma membrane of Neurospora crassa contains a proton-translocating ATPase, which functions to generate a large membrane potential and thereby to drive a variety of H+-dependent co-transport systems. We have purified this ATPase by a three-step procedure in which 1) loosely bound membrane proteins are removed by treatment with 0.1% deoxycholate; 2) the ATPase is solubilized with 0.6% deoxycholate in the presence of 45% glycerol; and 3) the solubilized enzyme is purified by centrifugation through a glycerol gradient. This procedure typically yields approximately 30% of the starting ATPase activity in a nearly homogeneous enzyme preparation of high specific activity, 61-98 mumol/min/mg of protein. The membrane-bound and purified forms of the ATPase are very similar with respect to kinetic properties (pH optimum, nucleotide and divalent cation specificity, sigmoid dependence upon Mg-ATP concentration) and sensitivity to inhibitors (including N,N'-dicyclohexylcarbodiimide and vanadate). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified ATPase displays a single major polypeptide band of Mr = 104,000, which is essentially identical in its electrophoretic mobility with the large subunit of [Na+, K+]-ATPase of animal cell membranes and [Ca2+]-ATPase of sarcoplasmic reticulum. The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.
The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO(3) with an estimated K(i) of 10 millimolar. The specific activity of the KNO(3)-sensitive ATPase was increased 29-fold during purification. N,N'-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl(-) and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins.
Membranes of Escherichia coli contain an adenosine 5'-triphosphate (ATP) energy-transducing system that is inhibited by treatment with dicyclohexylcarbodiimide (DCCD). The carbodiimide-reactive protein component of this system has been identified after treatment with [14C]DCCD. This protein has an apparent molecular weight of 9,000 as judged from acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is extracted from the membrane with chloroform-methanol (2:1). These properties are similar to the analogous protein previously identified in mitochondria (Cattell et al., 1971). A mutant strain, RF-7, has been isolated which derives energy from oxidative phosphorylation in the presence of 5 mM DCCD. The ATP hydrolase activity of the membraned system in the mutant was considerably less sensitive to inhibition by DCCD than that in the wild type. The carbodiimide-reactive protein, which was easily labeled by [14C]DCCD in the wild type, was labeled much less rapidly in the carbodiimide-resistant mutant. It is thus concluded that the reaction of DCCD with this specific protein leads to inhibition of the ATP energy-transducing reactions. The mutation causing carbodiimide resistance in strain RF-7 was mapped. It is cotransduced with the uncA gene at a frequency exceeding 90%. The mutationally altered protein causing the carbodiimide resistance was not conclusively identified. However, reconstitution experiments indicate that the altered protein is not one of the subunits of the soluble ATP hydrolase activity, which can be removed from the membrane by washing with 1 mM tris(hydroxymethyl)aminomethane buffer lacking Mg2+. The carbodiimide-reactive protein remains with the membrane residue after removal of the soluble ATP hydrolase and is thus distinct from these subunits as well.
This chapter presents a comprehensive list of compounds that inhibit the synthesis of adenosine triphosphate (ATP) associated with electron transport that is catalyzed by membrane systems present in mitochondria, chloroplasts, and prokaryotic cells. Inhibitors are divided into two groups. Some inhibitors interact directly with the ATPase molecule. In this group, the aurovertins, citreoviridin, Nbf-C1 (4-chloro-7-nitrobenzofurazan), quercetin, tentoxin, and efrapeptin are included. These compounds also react with and inhibit purified soluble ATPase preparations Other inhibitors react with other membrane components of the synthetase complex. These include carbodiimides, oligomycins, venturicidins, organotins, ossamycin, leucinostatin, and Dio-9. Some compounds whose inhibitory effects have not been well characterized are also discussed in the chapter. The chapter discusses the significant physical and chemical properties of these inhibitors.
A simple, rapid, and precise method is described for the continuous automated determination of the activity of membrane-bound enzymes which deliberate inorganic phosphate, e.g., ATPases and phosphatases. The characteristics of this method, which is based on the determination of liberated phosphate in the presence of nucleotides, are: (A) the enzyme reaction can be followed continuously during a certain period, thus providing a higher precision, as compared to other methods in which the enzyme reaction is measured by few distinct determinations; (B) the enzyme protein and other (membrane) proteins of the enzyme preparation have not to be removed during the continuous determination of enzyme activity because they remain solubilized after denaturation; and (C) low or moderate concentration of nonionic detergents do not disturb the reading of the absorbancy.
The presence of nonionic and cationic detergents interfered in the Lowry method of protein estimation by causing precipitate formation. The addition of 0.5% sodium dodecylsulphate in the alkali reagent prevented this precipitation without affecting colour development, and allowed the method to be used on detergent treated membrane preparations.
The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
This chapter describes the preparation of membrane fractions highly enriched for each of the ATPases, and the assay procedures used to quantitate the amount of activity specifically due to each enzyme. Three H+-translocating ATPases have been described in membranes of Neurospora crassa. The mitochondrial ATPase catalyzes the synthesis of ATP and has a complex structure consisting of 11 different polypetides, typical of the family of F0F1-ATPases. The plasma membrane ATPase serves as an electrogenic pump, hydrolyzing ATP to generate an electrochemical proton gradient, which can be used to drive a series of H+ cotransport systems. The vacuolar ATPase hydrolyzes ATP and generates a pH gradient (and possibly a membrane potential) that is coupled to the uptake of basic amino acids into the vacuolar interior. The primary purpose in isolating membranes from Neurospora has been to characterize the H+-translocating ATPases. Therefore, it has been important to obtain homogeneous preparations with a single type of ATPase. After solubilization, the ATPases tend to copurify, making mixed-membrane preparations unsuitable for enzyme purification. The most straightforward procedure for distinguishing among the three proton pumps is to measure their ATPase activities. The three membrane ATPases are characterized by different pH optima and specific inhibitor sensitivities.
This chapter discusses the preparation and reconstitution of F1F0 and F0 from Escherichia coli. The ATP synthase, F1F0, is extracted from everted membrane vesicles of E. coli by detergent and purified by ion-exchange chromatography and precipitation with polyethylene glycol (PEG). The proton conductor, F0, is isolated from F1F0 by dissociation of F1. The enzymatic activities of F1F0 assayed are ATPase, ATP-32Pi exchange, and ATP-dependent quenching of acridine dye fluorescence. F0 is tested for proton conduction by direct pH electrode measurements and by quenching of acridine dye fluorescence. These activities are inhibited by dicyclohexylcarbodiimide (DCCD). The easiest assay is the assay of ATPase activity, as it is not disturbed by detergents as long as some residual phospholipid is present. The other activities are dependent on a vesicular structure, so proteoliposomes have to be reconstituted from the enzyme preparation and phospholipids.
This chapter discusses the preparation, dissociation into subunits, and reconstitution of an active complex for the proton-conducting portion (F0) from Escherichia coli ATP synthase. The F0 part of the ATP synthase complex (F1F0) of E. coli is composed of three different polypeptides and the complex serves as a proton channel. After reconstitution into liposomes a functional F0 complex can be tested either by measuring passive proton translocation or by adding back F1. Passive proton translocation through F0 is measured using K+-loaded liposomes. ATPase activity of reconstituted F1F0 complexes is measured by determining the liberated orthophosphate. The chapter outlines the procedure for preparation of F0 by hydrophobic interaction chromatography. Reconstitution of a functional F0 complex is only achieved by incorporation of all three kinds of subunits into phospholipid vesicles.
During growth with low levels of K+, Bacillus acidocaldarius expressed a high-affinity K+ uptake system. The following observations indicate that this system strongly resembles the Kdp-ATPase of Escherichia coli: (i) its high affinity for K+ (Km of 20 microM or below); (ii) its poor transport of Rb+; (iii) the enhanced ATPase activity of membranes derived from cells grown with low levels of K+ (this activity was stimulated by K+ and inhibited by vanadate); (iv) the expression of an extra protein with a molecular weight of 70,000 in cells grown with low levels of K+; and (v) the immunological cross-reactivity of this 70,000-molecular-weight protein with antibodies against the catalytic subunit B of the E. coli Kdp system. Antibodies against the complete E. coli Kdp system, which immunoprecipitated the whole E. coli KdpABC complex, almost exclusively precipitated the 70,000-molecular-weight protein from detergent-solubilized B. acidocaldarius membranes. The possibility that the B. acidocaldarius Kdp system consists of a single, KdpB-type subunit is discussed.
This chapter covers the description of the procedures for the purification and the phosphorylation of the Kdp-ATPase. The Kdp-ATPase is one of at least two systems involved in potassium (K⁺) transport in Escherichia coli. The Kdp complex consists of three polypeptides located in the cytoplasmic membrane and is responsible for the K⁺-stimulated ATPase activity, associated with everted membrane vesicles. The formation of a phosphorylated intermediate during ATP hydrolysis suggests a functional homology to other eukaryotic ion transport ATPases. Frozen cells are thawed, washed once, and disrupted by a single passage through a Ribi cell fractionator (Sorvall). In the first step of the purification procedure, the everted vesicles are washed twice with lowionic-strength buffer, to remove peripherally membrane-associated proteins. The protein yield and specific activities of the Kdp-ATPase at the individual purification steps are summarized in the chapter. The washing of membranes with EDTA at low ionic strength proved to be an efficient initial step, removing about 40% of the peripheral or adsorbed membrane proteins. Under solubilization conditions, Aminoxid WS 35 extracted 50% of the residual membrane-bound proteins.
The purification and properties of a Na+- and K+-activated ATPase from ox brain is described. The enzyme preparation is characterized by a high purity and a low content (<1%) of Mg++-stimulated ATPase.
1.1. A procedure is presented that allows preparation of with specific activities of (units refer to μmoles ATP hydrolysed per min) by incubation of a microsomal fraction with sodium dodecylsulphate and ATP followed by a single zonal centrifugation.2.2. In a technically simple version of this procedure preparations with specific activities of 20–26 units · mg−1 protein are obtained in quantities of 12–20 mg protein by a single centrifugation, in an angle rotor, of microsomal fractions from the outer medulla of rabbit, pig or sheep kidneys.3.3. Binding of ATP and ADP partially protects against inactivation by sodium dodceylsulphate. In the incubation with sodium dodecylsulphate and ATP, 80% of the protein in the microsomal fraction is solubilized whereas the remains bound to membrane fragments. Tracer analysis shows that sodium dodecylsulphate forms less than 0.5% of the mass of the purified preparations.4.4. Analysis of the protein composition by sodium dodecylsulphate gel electrophoresis and determination of the capacities for binding of ATP and ouabain and for sodium-dependent phosphorylation (Jørgensen, P. L. (1974) Biochim. Biophys. Acta 356, 53–67 show that the procedures lead to a true purification of the enzyme.
The maximum yield for the production of L-681,110 by Streptomyces sp. MA-5038 (ATCC 31587) was observed after 5 days' incubation at 28 degrees C and pH about 8.3. L-681,110 was isolated from the fermentation broth by acetone extraction of the mycelia, absorption to Amberlite XAD-2 resin and two separations by thin-layer chromatography. The structure of L-681,110 was found to consist of a sixteen-membered lactone with a new type of substitution. The inhibition of ATPase, activity against Caenorhabditis elegans and stimulation of gamma-aminobutyric acid release indicate that L-681,110 possesses some characteristics of both oligomycin and avermectin. L-681,110 was also active against tapeworm and ticks in an in vivo assay.
This chapter presents the evidence obtained at different levels of biological organization that characterizes the plasma membrane ATPase of fungi and plants as a novel type of proton pump. It presents a parallel discussion of studies with fungal and plant cells to stress the similarities of their bioenergetic mechanisms that plant physiologists and microbial biochemists may have failed to appreciate. The membranes of bacteria, mitochondria, and chloroplasts contain a similar ATPase involved in proton transport. These enzymes are sensitive to azide and dicyclohexylcarbodiimide and, in the case of mitochondria and photosynthetic bacteria, to oligomycin. They do not form phosphorylated intermediates and they have alkaline pH optima and very complicated structures. These enzymes have a hydrophilic catalytic part with five different subunits protruding from the membrane and a hydrophobic part embedded in the membrane with three different subunits that form a proton channel blocked by dicyclohexyl-carbodiimide. A different type of ATPase is found in the microsomes and plasma membranes of animal cells. These enzymes catalyze the exchange of sodium for potassium and of protons for potassium and calcium transport. The ATPase activities are dependent on the presence of the transported cations and the pH optima are neutral.
The bafilomycins A1, A2, B1, B2, C1 and C2, a new type of macrolide antibiotics with a 16-membered lactone ring, were isolated from the fermentation broth of three Streptomyces griseus strains (TU 1922, TU 2437, TU 2599) by ethyl acetate extraction and column chromatography on silica gel. The bafilomycins exhibit activity against Gram-positive bacteria and fungi. Physico-chemical data, chemical structures and biological activities are reported.
Recent developments concerning the structure and function of the Ca2+ pump protein of the sarcoplasmic reticulum have been briefly reviewed. Various new methods have become available that make it possible to monitor dynamic changes in the structure of the enzyme molecule associated with elementary steps of the enzyme reaction. In the light of information about chemical reactivity of various amino acid residues and their location in the primary structure of the ATPase polypeptide, it will be fruitful to use extrinsic conformational probes placed at specific locations to monitor the kinetics of the enzyme. Furthermore, a growing body of evidence suggests that subunit-subunit interactions of an oligomeric Ca2+ ATPase are involved in the regulation of the kinetics of the enzyme. Thus the kinetic mechanisms has to be reinterpreted at all levels--i.e. primary, secondary, tertiary, and quaternary--of structure.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
We report here a new mode of ATP synthesis in living cells. The anaerobic bacterium Propionigenium modestum gains its total energy for growth from the conversion of succinate to propionate according to: succinate + H(2)O --> propionate + HCO(3) ( big up tri, openG' = -20.6 kJ/mol). The small free energy change of this reaction does not allow a substrate-linked phosphorylation mechanism, and no electron transport phosphorylation takes place. Succinate was degraded by cell-free extracts to propionate and CO(2) via succinyl-CoA, methyl-malonyl-CoA and propionyl-CoA. This pathway involves a membrane-bound methylmalonyl-CoA decarboxylase which couples the exergonic decarboxylation with a Na ion transport across the membrane. The organism also contained a membrane-bound ATPase which was specifically activated by Na ions and catalyzed and transport of Na ions into inverted bacterial vesicles upon ATP hydrolysis. The transport was abolished by monensin but not by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone. Isolated membrane vesicles catalyzed the synthesis of ATP from ADP and inorganic phosphate when malonyl-CoA was decarboxylated and malonyl-CoA synthesis from acetyl-CoA when ATP was hydrolyzed. These syntheses were sensitive to monensin which indicates that Na functions as the coupling ion. We conclude from these results that ATP synthesis in P. modestum is driven by a Na ion gradient which is generated upon decarboxylation of methylmalonyl-CoA.
- Y.-Z Wang
- H Sze
Wang, Y.-Z. & Sze, H. (1985) J. Biol. Chem. 260, 10434-10443.