Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins.     

Complex target SELEX

Aptamers are non-naturally occurring structured oligonucleotides that may bind to small molecules, peptides, and proteins. Typically, aptamers are generated by an in vitro selection process referred to as SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind with high affinity and specificity to proteins that reside on the cell surface have potential utility as therapeutic antagonists, agonists, and diagnostic agents. When the target protein requires the presence of the cell membrane (e.g., G-protein-coupled receptors, ion channels) or a co-receptor to fold properly, it is difficult or impossible to program the SELEX experiment with purified, soluble protein target. Recent advances in which the useful range of SELEX has been extended from comparatively simple purified forms of soluble proteins to complex mixtures of proteins in membrane preparations or in situ on the surfaces of living cells offer the potential to discover aptamers against previously intractable targets. Additionally, in cases in which a cell-type specific diagnostic is sought, the most desirable target on the cell surface may not be known. Successful application of aptamer selection techniques to complex protein mixtures can be performed even in the absence of detailed target knowledge and characterization. This Account presents a review of recent work in which membrane preparations or whole cells have been utilized to generate aptamers to cell surface targets. SELEX experiments utilizing a range of target "scaffolds" are described, including cell fragments, parasites and bacteria, viruses, and a variety of human cell types including adult mesenchymal stem cells and tumor lines. Complex target SELEX can enable isolation of potent and selective aptamers directed against a variety of cell-surface proteins, including receptors and markers of cellular differentiation, as well as determinants of disease in pathogenic organisms, and as such should have wide therapeutic and diagnostic utility.     

Does fusion of domains from unrelated proteins affect their folding pathways and the structural changes involved in their function? A case study with the diphtheria toxin T domain

We investigated whether the structural and functional behaviorsof two unrelated protein domains were modified when fused. TheIgG-binding protein ZZ derived from staphylococcal protein Awas fused to the N- and/or C-terminus of the diphtheria toxintransmembrane domain (T). T undergoes a conformational changefrom a soluble native state at neutral pH to a molten globule-likestate at acidic pH, leading to its interaction with membranes.We found that this molten globule state was not connected tothe GdnHCl-induced unfolding pathway of T. The pH-induced transitionof T, and also the unfolding of T and ZZ at neutral and acidicpH, were unchanged whether the domains were isolated or fused.The position of ZZ, however, influenced the solubility of Tnear its pK_i. SPR measurements revealed that T has a high affinityfor membranes, isolated or within the fusion proteins (K_D<10^-11 M). This work shows that in the case of T and ZZ, thefusion of protein domains with different stabilities does notalter the structural changes involved in folding and function.This supports the use of T as a soluble membrane anchor.     

Attachment of Cancer Urothelial Cells to the Bladder Epithelium Occurs on Uroplakin-Negative Cells and Is Mediated by Desmosomal and Not by Classical Cadherins

Urinary bladder cancer is often multifocal; however, the intraluminal dissemination of the urothelial cancer cells is poorly understood. The involvement of N-cadherin in the adhesion of the cancer urothelial cells to the urothelium had not previously been studied. Therefore, we herein explore the possibility of the intraluminal dissemination of the urothelial cancer cells by evaluating the role of classical cadherins in the adhesion of urothelial cancer cells to the urothelium. We used E-cadherin negative T24 cells and established a T24 Ncad^low cell line with an additionally decreased expression of N-cadherin in the plasma membrane and a decreased secretion of proform of metalloproteinase 2. The labelled T24 and T24 Ncad^low cells were seeded onto urothelial in vitro models. After 24 h in co-culture, unattached cancer cells were rinsed and urothelia with attached cancer urothelial cells were processed for fluorescence and electron microscopy. Both the T24 and T24 Ncad^low cells attached to the urothelium, yet only to the uroplakin-negative urothelial cells. The ultrastructural analysis showed that T24 and T24 Ncad^low cells adhere to poorly differentiated urothelial cells by desmosomes. To achieve this, they first disrupt tight junctions of superficial urothelial cells. This study indicates that the lack of E-cadherin expression and decreased expression of N-cadherin in the plasma membrane of T24 cells does not interfere with their adhesion to the urothelium; therefore, our results suggest that intraluminal dissemination of cancer urothelial cells along the urothelium occurs on uroplakin-negative cells and is desmosome-mediated.     

Hydrolysis Activity of Pyloric Cecal Enterocytes of Brown Trout (Salmo trutta) toward Monoacylglycerol and Lysophosphatidylcholine

Some lipid digestion pathways in fish deviate from those in mammals, and many differences may also be species dependent. This report describes a pathway for monoacylglycerol (MAG) and lysophospholipid absorption by intestinal enterocytes in brown trout that may be of significance in salmonids. When culturing primary cells in a medium containing 1‐ and 2‐MAG, we observed a massive hydrolysis of unesterified fatty acids. The hydrolysis activity was retained in the medium even after the removal of the cells. To further characterize these activities, both extracellular and isolated membrane proteins were tested for lipase activity toward triacylglycerol (TAG), diacylglycerol (DAG), MAG, phosphatidylcholine (PtdCho), and lysoPtdCho. In both cases, the main hydrolyzing activity was toward MAG followed by lysoPtdCho with very little activity toward DAG, TAG, or PtdCho. The extracellular and membrane proteins were partially purified by fast protein liquid chromatography and identified by proteomics (liquid chromatography–tandem mass spectrometry) focusing on lipase/hydrolase enzymes. In the membrane protein fraction, the data suggested that MAG was produced as an intermediate in the hydrolysis of lysoPtdCho by either lysophospholipase C or lysophospholipase D activity. Both abhydrolase‐domain‐containing protein 6 and abhydrolase‐domain‐containing protein 12 were identified in the membrane protein and they could be responsible for the hydrolysis of MAG. In the culture medium, low‐peptide matches were found for ABHD6 and phospholipases and further studies are needed. In summary, trout enterocytes are capable of hydrolyzing MAG and lysoPtdCho. The enzymes are both extracellular and membrane bound. The pathways may be of significance during lipid absorption in fish lacking a 1,3 specific pancreatic lipase.     

Insulin Induces Phosphorylation of Serine Residues of Translationally Controlled Tumor Protein in 293T Cells

Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.     

Redesigning an integral membrane K+ channel into a soluble protein

Even though the structure determination of soluble proteins has become routine, the number of unrelated integral membrane protein structures remains at a few dozen. The importance of this class of proteins to the molecular mechanisms underlying numerous biological phenomena demands that novel experimental techniques be developed to overcome the limitations imposed by conventional detergent-dependent approaches. Here we report the re-engineering of a putative K+ channel protein of unknown structure into a water-soluble analogue. By analyzing evolutionary conservation patterns of related sequences, lipid-facing residues of the primitive channel were identified and mutagenized into more polar alternatives. Further stabilization of the resultant construct was achieved through fusion with maltose-binding protein. The final soluble protein forms a tetramer, suggesting that it accurately models its predecessor. This methodology, as a viable alternative to the use of detergents, should be applicable to a wide range of integral membrane protein families including transporters and other signal transducers.     

Insight into Bortezomib Focusing on Its Efficacy against P-gp-Positive MDR Leukemia Cells

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.     

Tunicamycin depresses p-glycoprotein glycosylation without an effect on its membrane localization and drug efflux activity in l1210 cells

P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump.     

Targeting of Proteins for Translocation at the Endoplasmic Reticulum

The endoplasmic reticulum represents the gateway to the secretory pathway. Here, proteins destined for secretion, as well as soluble and membrane proteins that reside in the endomembrane system and plasma membrane, are triaged from proteins that will remain in the cytosol or be targeted to other cellular organelles. This process requires the faithful recognition of specific targeting signals and subsequent delivery mechanisms to then target them to the translocases present at the ER membrane, which can either translocate them into the ER lumen or insert them into the lipid bilayer. This review focuses on the current understanding of the first step in this process representing the targeting phase. Targeting is typically mediated by cleavable N-terminal hydrophobic signal sequences or internal membrane anchor sequences; these can either be captured co-translationally at the ribosome or recognised post-translationally and then delivered to the ER translocases. Location and features of the targeting sequence dictate which of several overlapping targeting pathway substrates will be used. Mutations in the targeting machinery or targeting signals can be linked to diseases.     

Do Extracellular Vesicles Derived from Mesenchymal Stem Cells Contain Functional Mitochondria?

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV.     

Structural Requirements for the Binding of a Peptide to Prohibitins on the Cell Surface of Monocytes/Macrophages

The screening of phage peptide libraries resulted in the identification of a sequence (named NW peptide, NWYLPWLGTNDW) that specifically binds to human monocytes and macrophages. Although the NW peptide can be used for the targeted delivery of therapeutics without knowledge of its receptor(s), the identification of-its binding partners will support future clinical applications-Here, we used the biotinylated NW peptide for cross-linking cell surface receptor(s) on live cells or as bait in pull-down assays with membrane proteins isolated from monocytes or human THP-1 cells differentiated into macrophages. Proteomic analysis of the captured proteins identified cell surface prohibitins (PHB1 and PHB2) and modified albumin as binding partners. Using flow cytometry and pull-down methods, we demonstrated that PHB1 and PHB2 interact directly with the NW peptide. Confocal imaging showed co-localization of the peptide with PHB1 on the surface of monocytes. Single replacement of either tryptophan or leucine with alanine completely inhibited binding, whereas the replacement of asparagine at position 1 or 10 and aspartic acid at position 11 with alanine did not affect the binding of the peptide variants. Neutral amino acid replacement of tryptophan at positions 2, 6, and 12 with tyrosine or phenylalanine also abolished the binding, implying that the indole ring of tryptophan is indispensable for the NW peptide to bind. Overall, the data suggest that membrane-associated prohibitins might be a useful target for the delivery of therapeutics to monocytes/macrophages and that tryptophan and leucine are key residues for peptide binding.     

The relationship of biochemical and morphological information in the central nervous system: The problem of sampling

The histology of the central nervous system (CNS) is complex, consisting of membranes derived from a number of cell types as well as specialized membranes associated with structural modifications of the neuron. The cell structure, and thus the membrane content, of the CNS varies with age and from area to area; in no instance is it possible to isolate a single cell type without special microdissection techniques. Neurological disease often involves specific areas of the CNS attacking one or all of the cellular elements in that region. In other instances, damage may be wide-spread, but at the histological level it may be restricted to a single cell type or even to a single membrane. The latter situation is particularly applicable to diseases that attack the myelin sheath. The biochemical investigation of changes in the lipid content of the brain resulting from neurological disease is hampered not only by the morphological complexity of nervous tissue, but by the tendency of destructive processes to be accompanied by proliferation of other cellular elements, both during the acute phase of disease and during the process of repair, which may mask significant abnormalities. Thus, in order to decide which changes in the structural lipids of the diseased brain are meaningful, a knowledge of the histopathology of the disorder is essential. When the morphological changes that accompany a neurological disorder are known, one may choose an appropriate tissue sample for study. Isolation of specific structural or functional subunits of the CNS, dissection of small groups of cells, isolation of single cells and separation of specific membranes are techniques available to sample nervous tissue. These are discussed, and their applicability to the biochemical study of specific neurological diseases is evaluated.     

Isolation of anti-T cell receptor scFv mutants by yeast surface display

Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.      

Review of the treatment of seafood processing wastewaters and recovery of proteins therein by membrane separation processes — prospects of the ultrafiltration of wastewaters from the fish meal industry

The wastewaters generated in fish meal production (average flow rates of 1100 m³/h for a plant capacity of 100 ton fish/h) contain a high organic load, and therefore they should not be discharged directly into the sea without effective treatment in order to prevent a negative impact on the environment. On the other hand, these effluents contain a large amount of potentially valuable proteins. These proteins can be concentrated by means of ultrafiltration (UF) and recycled into the fish meal process, improving its quality and the economic benefits from the raw material, whereas the treated water can be discharged into the sea or reused in the plant. Due to the high concentration of suspended matter in these effluents, microfiltration (MF) pre-treatment is required. An extensive review of the application of pressure-driven membrane separation processes in the treatment of seafood processing effluents and recovery of proteins therein is presented. Two effluents from a fish meal plant located in Talcahuano, Chile, were characterised and microfiltrated with a Whatman filter No. 1. A mineral tubular membrane, Carbosep M2 (MWCO = 15 kDa, ID = 6 mm and L = 1.2 m) was used in the UF experiments. The operating conditions were optimised in total recirculation mode, and the subsequent concentration experiments were carried out at 4 bar, 4 m/s, ambient temperature and natural pH. The results show that UF reduces the organic load from the fish meal wastewaters and allows the recovery of valuable raw materials comprising proteins. Moreover, they point out that further investigation should be dedicated to the use of UF membranes of lower molecular weight cut-off — or even NF membranes — in order to achieve complete recovery of the proteins contained in these effluents. Although the membrane undergoes severe fouling, it can easily be cleaned through a caustic washing.     

In vitro toxicoproteomic analysis of A549 human lung epithelial cells exposed to urban air particulate matter and its water-soluble and insoluble fractions

Background

Toxicity of airborne particulate matter (PM) is difficult to assess because PM composition is complex and variable due to source contribution and atmospheric transformation. In this study, we used an in vitro toxicoproteomic approach to identify the toxicity mechanisms associated with different subfractions of Ottawa urban dust (EHC-93).

Methods

A549 human lung epithelial cells were exposed to 0, 60, 140 and 200 μg/cm² doses of EHC-93 (total), its insoluble and soluble fractions for 24 h. Multiple cytotoxicity assays and proteomic analyses were used to assess particle toxicity in the exposed cells.

Results

The cytotoxicity data based on cellular ATP, BrdU incorporation and LDH leakage indicated that the insoluble, but not the soluble, fraction is responsible for the toxicity of EHC-93 in A549 cells. Two-dimensional gel electrophoresis results revealed that the expressions of 206 protein spots were significantly altered after particle exposures, where 154 were identified by MALDI-TOF-TOF-MS/MS. The results from cytotoxicity assays and proteomic analyses converged to a similar finding that the effects of the total and insoluble fraction may be alike, but their effects were distinguishable, and their effects were significantly different from the soluble fraction. Furthermore, the toxic potency of EHC-93 total is not equal to the sum of its insoluble and soluble fractions, implying inter-component interactions between insoluble and soluble materials resulting in synergistic or antagonistic cytotoxic effects. Pathway analysis based on the low toxicity dose (60 μg/cm²) indicated that the two subfractions can alter the expression of those proteins involved in pathways including cell death, cell proliferation and inflammatory response in a distinguishable manner. For example, the insoluble and soluble fractions differentially affected the secretion of pro-inflammatory cytokines such as MCP-1 and IL-8 and distinctly altered the expression of those proteins (e.g., TREM1, PDIA3 and ENO1) involved in an inflammatory response pathway in A549 cells.

Conclusions

This study demonstrated the impact of different fractions of urban air particles constituted of various chemical species on different mechanistic pathways and thus on cytotoxicity effects. In vitro toxicoproteomics can be a valuable tool in mapping these differences in air pollutant exposure-related toxicity mechanisms.

     

Insights into the Role of the Discontinuous TM7 Helix of Human Ferroportin through the Prism of the Asp325 Residue

The negatively charged Asp325 residue has proved to be essential for iron export by human (HsFPN1) and primate Philippine tarsier (TsFpn) ferroportin, but its exact role during the iron transport cycle is still to be elucidated. It has been posited as being functionally equivalent to the metal ion-coordinating residue His261 in the C-lobe of the bacterial homolog BbFpn, but the two residues arise in different sequence motifs of the discontinuous TM7 transmembrane helix. Furthermore, BbFpn is not subject to extracellular regulation, contrary to its mammalian orthologues which are downregulated by hepcidin. To get further insight into the molecular mechanisms related to iron export in mammals in which Asp325 is involved, we investigated the behavior of the Asp325Ala, Asp325His, and Asp325Asn mutants in transiently transfected HEK293T cells, and performed a comparative structural analysis. Our biochemical studies clearly distinguished between the Asp325Ala and Asp325His mutants, which result in a dramatic decrease in plasma membrane expression of FPN1, and the Asp325Asn mutant, which alters iron egress without affecting protein localization. Analysis of the 3D structures of HsFPN1 and TsFpn in the outward-facing (OF) state indicated that Asp325 does not interact directly with metal ions but is involved in the modulation of Cys326 metal-binding capacity. Moreover, models of the architecture of mammalian proteins in the inward-facing (IF) state suggested that Asp325 may form an inter-lobe salt-bridge with Arg40 (TM1) when not interacting with Cys326. These findings allow to suggest that Asp325 may be important for fine-tuning iron recognition in the C-lobe, as well as for local structural changes during the IF-to-OF transition at the extracellular gate level. Inability to form a salt-bridge between TM1 and TM7b during iron translocation could lead to protein instability, as shown by the Asp325Ala and Asp325His mutants.     

Genomic and Targeted Approaches Unveil the Cell Membrane as a Major Target of the Antifungal Cytotoxin Amantelide A

Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.