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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Programmed hepatocytes cell death associated with FLIP downregulation in response to extracellular preS1/2.

Chronic hepatitis B virus (HBV) infection involves liver damage resulting in continuous cell injury and death. During HBV infection, hepatocytes exhibit changes in death receptor expression and in their susceptibility to death. These changes are observed not only in infected cells but also in bystander cells. Because excess viral surface protein (HBsAg) is secreted in large amounts as soluble particles containing preS proteins, the role of soluble preS1/2 in hepatocyte (HepG2) death modulation is an important issue to be explored. An increase of cell death induced by preS1/2 was observed. Also, cell death was associated with the down-regulation of FLIP and activation of caspase 8, caspase 9, and BID. Additionally, hepatocytes exhibited a sensitization to death mediated by the Fas receptor. These results, may contribute to understanding the role of envelope proteins (preS1/2) in the pathogenesis of HBV infection.

Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein.

Hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are involved in the first steps of virus infection. The E2 ectodomain can be produced as an isolated form (E2661). However, there is some concern about its proper conformation and the role that E1 can play as a chaperone for the folding of E2. In order to verify this fact we have expressed a chimeric protein (E1tmbE2) based on the full-length E1 sequence followed by the E2 ectodomain using the baculovirus-insect cells system. The E2 ectodomain is folded in the presence of the E1, proteolytically processed by cellular proteases and secreted to cell culture media (E2661p), while the E1 protein is retained into the cell due to its transmembrane sequence. The purification of E2661p from culture media was facilitated by a His tag introduced in its amino terminus. Both E2661 and E2661p glycoproteins shared very similar structural features, monitored by spectroscopic and antigenic studies. Moreover, their functional properties, tested by means of CD81 binding, were almost indistinguishable, indicating that the E2 ectodomain constitutes an independent folding unit.

Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis.

OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces, which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR-DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.

A deficient translocation of CD3ζ, ZAP-70 and Grb2 to lipid raft, as a hallmark of defective adaptive immune response during chronic hepatitis B infection.

Hepatitis B is considered to be a worldwide public health problem. An immunosuppressor microenvironment has been proposed to contribute to viral persistence during chronic disease. Understanding the intracellular signaling cascade in T-cells from HBV-infected patients, will contribute to unravel the mechanisms that control the development of immune response during hepatitis B. We analyze lipid rafts formation and early activation signals in chronic HBV infected patients, compared to naturally immune subjects (NIS). Patients show: (1) diminished GM1 clustering, (2) A deficient lipid rafts recruitment of CD3ζ/ZAP-70/Grb2, and (3) these proteins do not merge with GM1 within the lipid rafts. Finally, immunoprecipitation assays proved that ZAP-70 does not associate to CD3ζ. These results show for the first time, defects regarding early key events in T-cell activation, in chronically infected HBV patients, which may contribute not only to understand HBV immune tolerance, but to reveal new potential therapeutic targets to control the infection.

Spectroscopic characterization and fusogenic properties of preS domains of duck hepatitis B virus.

In order to shed light on the hepatitis B virus fusion mechanism and to explore the fusogenic capabilities of preS regions, a recombinant duck hepatitis B virus (DHBV) preS protein (DpreS) containing six histidines at the carboxy-terminal end has been obtained. The DpreS domain, which has an open and mostly nonordered conformation as indicated by fluorescence and circular dichroism spectroscopies, has the ability to interact with negatively charged phospholipid vesicles. The observed interaction differences between neutral and acidic phospholipids can be interpreted in terms of an initial ionic interaction between the phospholipid polar headgroup and the protein followed by the insertion of probably the N-terminal region in the cellular membrane. Fluorescence polarization studies detect a decrease of the transition enthalpy together with a small modification of the transition temperature, typical effects of integral membrane proteins. The interaction of the protein with acidic phospholipid vesicles induces aggregation, lipid mixing, and leakage of internal contents, properties that have been ascribed to membrane destabilizing proteins. The fact that the preS domains of the hepadnaviruses have little similarity but share a very similar hydrophobic profile points to the importance of the overall three-dimensional structure as well as to its conformational flexibility and the distribution of polar and apolar amino acids on the expression of their destabilizing properties rather than to a particular amino acid sequence. The results presented herein argue for the involvement of DpreS in the initial steps of DHBV infection. Taken together with previously reported results, the conclusion that both S and preS regions participate in the fusion process of the hepadnaviridae family may be drawn.

Interaction of the hepatitis B core antigen and the innate immune system.

Previous studies demonstrated that the primary APCs for the hepatitis B core Ag (HBcAg) were B cells and not dendritic cells (DC). We now report that splenic B1a and B1b cells more efficiently present soluble HBcAg to naive CD4(+) T cells than splenic B2 cells. This was demonstrated by direct HBcAg-biotin-binding studies and by HBcAg-specific T cell activation in vitro in cultures of naive HBcAg-specific T cells and resting B cell subpopulations. The inability of DC to function as APCs for exogenous HBcAg relates to lack of uptake of HBcAg, not to processing or presentation, because HBcAg/anti-HBc immune complexes can be efficiently presented by DC. Furthermore, HBcAg-specific CD4(+) and CD8(+) T cell priming with DNA encoding HBcAg does not require B cell APCs. TLR activation, another innate immune response, was also examined. Full-length (HBcAg(183)), truncated (HBcAg(149)), and the nonparticulate HBeAg were screened for TLR stimulation via NF-kappaB activation in HEK293 cells expressing human TLRs. None of the HBc/HBeAgs activated human TLRs. Therefore, the HBc/HBeAg proteins are not ligands for human TLRs. However, the ssRNA contained within HBcAg(183) does function as a TLR-7 ligand, as demonstrated at the T and B cell levels in TLR-7 knockout mice. Bacterial, yeast, and mammalian ssRNA encapsidated within HBcAg(183) all function as TLR-7 ligands. These studies indicate that innate immune mechanisms bridge to and enhance the adaptive immune response to HBcAg and have important implications for the use of hepadnavirus core proteins as vaccine carrier platforms.

Improving on the ability of endogenous hepatitis B core antigen to prime cytotoxic T lymphocytes.

Hepatitis B virus core antigen (HBcAg) is thought to be a major target for specific cytotoxic T cells (CTLs) in hepatitis B virus infections. A single dose of hepatitis C virus nonstructural 3/4A DNA (<5 microg) effectively primes functional specific CTLs, independently of CD4(+) T helper cells and by different routes of immunization. In contrast, HBcAg-specific CTL priming was T helper cell dependent and highly sensitive to the dose and route of delivery. Although CTL priming was improved 10-fold by codon optimization and in vivo electroporation, low levels of DNA still failed to prime CTLs effectively. Only high doses (5 microg) of codon-optimized HBcAg delivered by in vivo electroporation primed in vivo lytic and polyfunctional CTLs. The ability of endogenous HBcAg to prime CTLs is surprisingly inefficient and differs from that of nonstructural 3/4A. This has important implications for the design of HBcAg-based therapeutic vaccines in humans.

Interaction of preS domains of hepatitis B virus with phospholipid vesicles.

The role of preS domains of the hepatitis B virus (HBV) envelope proteins in the first steps of viral infection has been restricted to their implication in virus attachment to a putative hepatocyte receptor. In order to explore a fusion activity in these regions, we used recombinant preS domains to characterize their interaction with liposomes. Binding experiments carried out with NBD-labeled proteins indicated that preS were able to interact in a monomeric way with acidic phospholipid vesicles, being the partition coefficient similar to that described for peptides which can insert deeply into bilayers. Fluorescence depolarization of DPH-labeled vesicles confirmed the specificity for negative charged phospholipids. Upon interaction the proteins induced aggregation, lipid mixing and release of internal contents of acidic vesicles at both acid and neutral pH in a concentration-dependent manner. Taken together, all these data indicate that preS domains are able to insert into the hydrophobic core of the bilayer. Moreover, the insertion resulted in a protein conformational change which increased the helical content. Therefore all these results suggest that, besides their participation in the recognition of a cellular receptor, the preS domains could be involved in the fusion mechanism of HBV with the plasma membrane of target cells.