Targeted dual degradation of HER2 and EGFR obliterates oncogenic signaling, overcomes therapy resistance, and inhibits metastatic lesions in HER2-positive breast cancer models.

AIMS: Human epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase amplified in approximately 20% of breast cancer (BC). HER2-targeted therapies are the linchpin of treating HER2-positive BC. However, drug resistance is common, and the main resistance mechanism is unknown. We tested the hypothesis that drug resistance results mainly from inadequate or lack of inhibition of HER2 and its family member epidermal growth factor receptor (EGFR). METHODS: We used clinically relevant cell and tumor models to assess the impact of targeted degradation of HER2 and EGFR on trastuzumab resistance. Trastuzumab is the most common clinically used HER2 inhibitor. Targeted degradation of HER2 and EGFR was achieved using recombinant human protein PEPDG278D, which binds to the extracellular domains of the receptors. siRNA knockdown was used to assess the relative importance of EGFR and HER2 in trastuzumab resistance. RESULTS: Both HER2 and EGFR are overexpressed in all trastuzumab-resistant HER2-positive BC cell and tumor models and that all trastuzumab-resistant models are highly vulnerable to targeted degradation of HER2 and EGFR. Degradation of HER2 and EGFR induced by PEPDG278D causes extensive inhibition of oncogenic signaling in trastuzumab-resistant HER2-positive BC cells. This is accompanied by strong growth inhibition of cultured cells, orthotopic patient-derived xenografts, and metastatic lesions in the brain and lung of trastuzumab-resistant HER2-positive BC. siRNA knockdown indicates that eliminating both HER2 and EGFR is necessary to maximize therapeutic outcome. CONCLUSIONS: This study unravels the therapeutic vulnerability of trastuzumab-resistant HER2-positive BC and shows that an agent that targets the degradation of both HER2 and EGFR is highly effective in overcoming drug resistance in this disease. The findings provide new insights and innovations for advancing treatment of drug-resistant HER2-positive breast cancer that remains an unmet problem.

Phage-Related Ribosomal Protease (Prp) of Staphylococcus aureus: In Vitro Michaelis-Menten Kinetics, Screening for Inhibitors, and Crystal Structure of a Covalent Inhibition Product Complex.

Phage-related ribosomal proteases (Prps) are essential for the assembly and maturation of the ribosome in Firmicutes, including the human pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Clostridium difficile. These bacterial proteases cleave off an N-terminal extension of a precursor of ribosomal protein L27, a processing step that is essential for the formation of functional ribosomes. This essential role of Prp in these pathogens has identified this protease as a potential antibiotic target. In this work, we determine the X-ray crystal structure of a covalent inhibition complex at 2.35 Å resolution, giving the first complete picture of the active site of a functional Prp. We also characterize the kinetic activity and screen for potential inhibitors of Prp. This work gives the most complete characterization of the structure and specificity of this novel class of proteases to date.

Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors.

Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.

Kinetics and inhibition studies of the L205R mutant of cAMP-dependent protein kinase involved in Cushing's syndrome.

Overproduction of cortisol by the hypothalamus-pituitary-adrenal hormone system results in the clinical disorder known as Cushing's syndrome. Genomics studies have identified a key mutation (L205R) in the α-isoform of the catalytic subunit of cAMP-dependent protein kinase (PKACα) in adrenal adenomas of patients with adrenocorticotropic hormone-independent Cushing's syndrome. Here, we conducted kinetics and inhibition studies on the L205R-PKACα mutant. We have found that the L205R mutation affects the kinetics of both Kemptide and ATP as substrates, decreasing the catalytic efficiency (kcat/KM) for each substrate by 12-fold and 4.5-fold, respectively. We have also determined the IC 50 and Ki for the peptide substrate-competitive inhibitor PKI(5-24) and the ATP-competitive inhibitor H89. The L205R mutation had no effect on the potency of H89, but causes a > 250-fold loss in potency for PKI(5-24). Collectively, these data provide insights for the development of L205R-PKACα inhibitors as potential therapeutics.

Fusogenic properties of the Ectodomain of HCV E2 envelope protein.

The steps leading from hepatitis C virus (HCV) attachment to the hepatocytes to the fusion of viral and cellular membranes remain uncharacterized. In this regard, we have studied the mechanism underlying the HCV fusion process using liposomes and a truncated form of E2 protein lacking the transmembrane region, E2661 (amino acids 384-661). E2661 has been previously obtained by using the baculovirus expression system and shown to behave as an independent folding domain (M. Rodriguez-Rodriguez, D. Tello, B. Yelamos, J. Gomez-Gutierrez, B. Pacheco, S. Ortega, A.G. Serrano, D.L. Peterson, F. Gavilanes, Structural properties of the ectodomain of hepatitis C virus E2 envelope protein, Virus Res. 139 (2009) 91-99). This form has been used in lipid-protein interaction studies with different model vesicles, at different pHs and by employing a variety of fluorescent assays. The obtained results indicate that E2661 induces vesicle aggregation, lipid mixing and liposome leakage, reaching higher values in the presence of negatively charged phospholipids and cholesterol at acidic pH. Therefore, the results of these studies would be indicative of an HCV infection process through receptor mediated endocytosis. Accordingly, E2 might be important in the HCV initial infective steps, interacting with the target membranes and giving rise to their subsequent destabilization.

Characterization of PKACα enzyme kinetics and inhibition in an HPLC assay with a chromophoric substrate.

Here we describe a convenient, inexpensive, and non-hazardous method for the measurement of the kinase activity of the catalytic subunit of cAMP-dependent protein kinase (PKACα). The assay is based on the separation of a substrate peptide labeled with a strong chromophore from the phosphorylated product peptide by high-performance liquid chromatograph (HPLC) and quantification of the product ratiometrically at a wavelength in the visual spectrum (Vis). The utility and reliability of the HPLC-Vis assay were demonstrated by characterizing the kinetic parameters (KM, Vmax) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC50, Ki) of the known PKACα inhibitors H89 and PKI(5-24). The advantages of this assay are that it is convenient and inexpensive, uses readily synthesized or commercially available substrates that are shelf-stable, uses a common piece of laboratory equipment, and does not require any hazardous materials such as radioactive γ-32P-ATP. The assay format is also highly flexible and could be adapted for the testing of many different kinases by changing the peptide substrate sequence.

Diseño de una prueba diagnóstica casera para la detección de anticuerpos tipo IgG contra el virus de la hepatitis C: incorporación de la proteína de envoltura E2 y no estructural 2 (NS2) / Design of an in-house diagnostic test for IgG antibodies against hepatitis C virus detection: Incorporation of envelope protein E2 and non-structural protein 2 ( NS2)

Durante la infección por el virus de la hepatitis C (VHC), los anticuerpos específicos aparecen varias semanas posterior a la exposición, van dirigidos contra las diversas proteínas del virus incluyendo anticuerpos contra la envoltura viral (E2)y la proteína no estructural 2 (NS2). En este trabajo se diseñó un ensayo casero de ELISA que incorpora, además de NS3, NS5a, NS5b y el core, a las proteínas E2 y NS2 del VHC en sustitución de NS4, con el fin de evaluar su capacidad diagnóstica en comparación con un estuche comercial de 4ta generación. La validación de la prueba casera demostró una especificidad y sensibilidad similar a las obtenidas con el estuche comercial de 4ta generación (Biokit©), con un índice kappa igual a 0,969, al compararse con el mismo. Esto sugiere que la prueba diseñada podría utilizarse de manera segura para la detección de anticuerpos VHC específicos de tipo IgG para el diagnóstico de la hepatitis C y constituirse como una alternativa de producción nacional más económica.

During hepatitis C (HCV) infection specific antibodies appear several weeks after exposure, including viral envelope (E2) and non-structural protein 2 ( NS2). In this work we designed an in-house ELISA assay, that incorporate beside NS3, NS5a, NS5b and core, the HCVE2 and NS2 proteins in substitution of NS4, in order to evaluate its diagnostic utility as compared to a fourth generation commercial kit. The in-house test demonstrated a specificity and sensitivity similar to those obtained with the commercial kit, with a kappa index equal to 0.969, when it was compared with the 4th generation commercial kit (BioelisaBiokit ©), suggesting that our test could be used for the diagnosis of specific IgG antibodies detection against hepatitis C and to become a more economic national alternative.

Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus.

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N-terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N-terminal extension also encode a sequence-specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un-cleavable N-terminal extension, or lacking the extension (pre-cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N-terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site-directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway.

Identification of Nef-HIV-1 domains involved in p22-phox interaction and superoxide production / Identificación del dominio de Nef-VIH-1 involucrado en la interacción con p22-phox y producción de superoxido

Nef -HIV-1 has been shown to be involved in NADPH complex interaction and superoxide production. The aim of this work was to study the domains involved in the interaction between Nef and p22-phox. Two approaches were used: 1) in silico modelling, to determine the potential binding motifs and design Nef truncated forms and 2) functional assays. The results showed that GFPVT 68-72, FPDW 121-124 and REVLE 179-183 on Nef are critical for p22-phox (RPQIG 142-146 and PGGP 181-184) docking. However, only the region containing FPDW 121-124 on Nef is able to induce superoxide production. Understanding the molecular mechanisms involved in generating oxidative stress during HIV infection, is critical for therapeutic intervention, in order to minimize viral replication and dissemination.

Se ha evidenciado que Nef-VIH-1 está involucrado en la interacción con el complejo NADPH y la producción de superóxido. El objetivo de este trabajo fue identificar los dominios implicados en la interacción entre Nef y p22-phox. Se utilizaron dos estrategias: 1) análisis in silico para determinar los posibles motivos de unión y el diseño Nef formas truncadas y 2) ensayos funcionales. Los resultados mostraron que GFPVT 68-72, FPDW 121 a 124 y 179 a 183 REVLE de Nef son críticos para su unión con p22-phox (RPQIG 142-146 y 181-184 PGGP). Sin embargo, sólo la región que contiene FPDW 121-124 en Nef, es capaz de inducir la producción de superóxido. La comprensión de los mecanismos moleculares implicados en la generación de estrés oxidativo durante la infección por VIH, es crítico para la intervención terapéutica, con el fin de minimizar la replicación y la propagación viral.

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection.

Design, synthesis, and in vitro evaluation of a fluorescently labeled irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKACα).

The design and development of irreversible kinase inhibitors is an expanding frontier of kinase drug discovery. The current approach to develop these inhibitors utilizes ATP-competitive inhibitor scaffolds to target non-catalytic cysteines in the kinase ATP-binding site. However, this approach is limited as not all kinases have a cysteine in the ATP-binding site that can be targeted. In this work, we report a complementary approach to developing irreversible kinase inhibitors that utilizes the substrate-binding site. Using the catalytic subunit of cAMP-dependent protein kinase (PKACα) as a model system, we have designed and synthesized an irreversible inhibitor based on the substrate-competitive inhibitor scaffold PKI(14-22) that covalently modifies non-catalytic Cys199 in the PKACα substrate-binding site. The new compound inhibits PKACα (IC50 = 11.8 ± 1.1 nM), is ∼100-fold selective for PKACα in a kinase panel, and covalently labels the kinase as demonstrated by fluorescence, mass spectrometry, and kinetics experiments. This study demonstrates the feasibility of utilizing this new approach to develop irreversible inhibitors for any of the eighty-nine kinases that possess a similar non-catalytic cysteine in their substrate-binding sites.

The deletion of residues 268-292 of E1 impairs the ability of HCV envelope proteins to induce pore formation.

We have obtained a chimeric protein containing the ectodomains of hepatitis C virus (HCV) envelope proteins but lacking the region 268-292 of E1. All its structural properties are coincident with those of the corresponding full length chimera. The deleted and entire chimeras were compared in terms of their membrane destabilizing properties. No differences were found in their ability to induce vesicle aggregation and lipid mixing but the deleted chimera showed a reduced capacity to promote leakage. The role of the deletion was also studied by obtaining HCV pseudoparticles (HCVpp). Both E1 and E2, and also the E1 deleted mutant, were incorporated into HCVpp to a similar level. However, HCVpp containing the E1 deleted protein are almost unable to infect Huh7 cells. These results point to the involvement of the region 268-292 in the formation of pores in the membrane necessary for the complete fusion of the membranes.

Dissecting the structural basis of MEIG1 interaction with PACRG.

The product of the meiosis-expressed gene 1 (MEIG1) is found in the cell bodies of spermatocytes and recruited to the manchette, a structure unique to elongating spermatids, by Parkin co-regulated gene (PACRG). This complex is essential for targeting cargo to the manchette during sperm flagellum assembly. Here we show that MEIG1 adopts a unique fold that provides a large surface for interacting with other proteins. We mutated 12 exposed and conserved amino acids and show that four of these mutations (W50A, K57E, F66A, Y68A) dramatically reduce binding to PACRG. These four amino acids form a contiguous hydrophobic patch on one end of the protein. Furthermore, each of these four mutations diminishes the ability of MEIG1 to stabilize PACRG when expressed in bacteria. Together these studies establish the unique structure and key interaction surface of MEIG1 and provide a framework to explore how MEIG1 recruits proteins to build the sperm tail.

Identification of Nef-HIV-1 domains involved in p22-phox interaction and superoxide production.

Nef -HIV-1 has been shown to be involved in NADPH complex interaction and superoxide production. The aim of this work was to study the domains involved in the interaction between Nef and p22-phox. Two approaches were used: 1) in silico modelling, to determine the potential binding motifs and design Nef truncated forms and 2) functional assays. The results showed that GFPVT 68-72, FPDW 121-124 and REVLE 179-183 on Nef are critical for p22-phox (RPQIG 142-146 and PGGP 181-184) docking. However, only the region containing FPDW 121-124 on Nef is able to induce superoxide production. Understanding the molecular mechanisms involved in generating oxidative stress during HIV infection, is critical for therapeutic intervention, in order to minimize viral replication and dissemination.

P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections.

In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x10(6)) and provided 80-100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.

Palivizumab epitope-displaying virus-like particles protect rodents from RSV challenge.

Respiratory syncytial virus (RSV) is the most common cause of serious viral bronchiolitis in infants, young children, and the elderly. Currently, there is not an FDA-approved vaccine available for RSV, though the mAb palivizumab is licensed to reduce the incidence of RSV disease in premature or at-risk infants. The palivizumab epitope is a well-characterized, approximately 24-aa helix-loop-helix structure on the RSV fusion (F) protein (F254-277). Here, we genetically inserted this epitope and multiple site variants of this epitope within a versatile woodchuck hepadnavirus core-based virus-like particle (WHcAg-VLP) to generate hybrid VLPs that each bears 240 copies of the RSV epitope in a highly immunogenic arrayed format. A challenge of such an epitope-focused approach is that to be effective, the conformational F254-277 epitope must elicit antibodies that recognize the intact virus. A number of hybrid VLPs containing RSV F254-277 were recognized by palivizumab in vitro and elicited high-titer and protective neutralizing antibody in rodents. Together, the results from this proof-of-principle study suggest that the WHcAg-VLP technology may be an applicable approach to eliciting a response to other structural epitopes.

A direct, ratiometric, and quantitative MALDI-MS assay for protein methyltransferases and acetyltransferases.

Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase.

Kinetic mechanism of protein N-terminal methyltransferase 1.

The protein N-terminal methyltransferase 1 (NTMT1) catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine to the protein α-amine, resulting in formation of S-adenosyl-l-homocysteine and α-N-methylated proteins. NTMT1 is an interesting potential anticancer target because it is overexpressed in gastrointestinal cancers and plays an important role in cell mitosis. To gain insight into the biochemical mechanism of NTMT1, we have characterized the kinetic mechanism of recombinant NTMT1 using a fluorescence assay and mass spectrometry. The results of initial velocity, product, and dead-end inhibition studies indicate that methylation by NTMT1 proceeds via a random sequential Bi Bi mechanism. In addition, our processivity studies demonstrate that NTMT1 proceeds via a distributive mechanism for multiple methylations. Together, our studies provide new knowledge about the kinetic mechanism of NTMT1 and lay the foundation for the development of mechanism-based inhibitors.

A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.

A key event in the process of spermiogenesis is the formation of the flagella, which enables sperm to reach eggs for fertilization. Yeast two-hybrid studies revealed that meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) interact, and that sperm-associated antigen 16, which encodes an axoneme central apparatus protein, is also a binding partner of MEIG1. In spermatocytes of wild-type mice, MEIG1 is expressed in the whole germ cell bodies, but the protein migrates to the manchette, a unique structure at the base of elongating spermatid that directs formation of the flagella. In the elongating spermatids of wild-type mice, PACRG colocalizes with α-tubulin, a marker for the manchette, whereas this localization was not changed in the few remaining elongating spermatids of Meig1-deficient mice. In addition, MEIG1 no longer localizes to the manchette in the remaining elongating spermatids of Pacrg-deficient mice, indicating that PACRG recruits MEIG1 to the manchette. PACRG is not stable in mammalian cells, but can be stabilized by MEIG1 or by inhibition of proteasome function. SPAG16L is present in the spermatocyte cytoplasm of wild-type mice, and in the manchette of elongating spermatids, but in the Meig1 or Pacrg-deficient mice, SPAG16L no longer localizes to the manchette. By contrast, MEIG1 and PACRG are still present in the manchette of Spag16L-deficient mice, indicating that SPAG16L is a downstream partner of these two proteins. Together, our studies demonstrate that MEIG1/PACRG forms a complex in the manchette and that this complex is necessary to transport cargos, such as SPAG16L, to build the sperm flagella.

Study of the putative fusion regions of the preS domain of hepatitis B virus.

In a previous study, it was shown that purified preS domains of hepatitis B virus (HBV) could interact with acidic phospholipid vesicles and induce aggregation, lipid mixing and leakage of internal contents which could be indicative of their involvement in the fusion of the viral and cellular membranes (Núñez, E. et al. 2009. Interaction of preS domains of hepatitis B virus with phospholipid vesicles. Biochim. Biophys. Acta 17884:417-424). In order to locate the region responsible for the fusogenic properties of preS, five mutant proteins have been obtained from the preS1 domain of HBV, in which 40 amino acids have been deleted from the sequence, with the starting point of each deletion moving 20 residues along the sequence. These proteins have been characterized by fluorescence and circular dichroism spectroscopy, establishing that, in all cases, they retain their mostly non-ordered conformation with a high percentage of ß structure typical of the full-length protein. All the mutants can insert into the lipid matrix of dimyristoylphosphatidylglycerol vesicles. Moreover, we have studied the interaction of the proteins with acidic phospholipid vesicles and each one produces, to a greater or lesser extent, the effects of destabilizing vesicles observed with the full-length preS domain. The ability of all mutants, which cover the complete sequence of preS1, to destabilize the phospholipid bilayers points to a three-dimensional structure and/or distribution of amino acids rather than to a particular amino acid sequence as being responsible for the membrane fusion process.