The events leading to the formation of β-amyloid (βA4) from its precursor (βAPP) involve proteolytic cleavages that produce the amino and carboxyl termini of βA4. The enzyme activities responsible for these cleavages have been termed β- and γ-secretase, respectively, although these protease(s) have not been identified. Since βA4 is known to possess heterogeneity at both the amino and carboxyl termini, β- and γ-secretases may actually be a collection of proteolytic activities or perhaps a single proteolytic enzyme with broad amino acid specificity.We investigated the role of cathepsin D in the processing of βAPP since this enzyme has been widely proposed as a γ-secretase candidate. Treatment of a synthetic peptide that spans the γ-secretase site of βAPP with human cathepsin D resulted in the cleavage of this substrate at Ala42-Thr43. A sensitive liquid chromatography/mass spectrometry technique was also developed to further investigate the ability of cathepsin D to process longer recombinant βAPP substrates (156 and 100 amino acids of βAPP carboxyl terminus) in vitro. The precise cathepsin D cleavage sites within these recombinant βAPP substrates were identified using this technique. Both recombinant substrates were cleaved at the following sites: Leu49-Val50, Asp68-Ala69, Phe93-Phe94. No cleavages were observed at putative γ-secretase sites: Val40-Ile41 or Ala42-Thr43, suggesting that cathepsin D is not γ-secretase as defined by these βA4 termini. Under conditions where the βAPP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of βA4, Glu−3-Val−2 and Glu3-Phe4, were observed, indicating that cathepsin D cleavage of βAPP is influenced by the structural integrity of the substrate. Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as γ-secretase; however, the ability of this enzyme to efficiently cleave βAPP substrates at nonamyloidogenic sites within the molecule may reflect a role in βAPP catabolism. The events leading to the formation of β-amyloid (βA4) from its precursor (βAPP) involve proteolytic cleavages that produce the amino and carboxyl termini of βA4. The enzyme activities responsible for these cleavages have been termed β- and γ-secretase, respectively, although these protease(s) have not been identified. Since βA4 is known to possess heterogeneity at both the amino and carboxyl termini, β- and γ-secretases may actually be a collection of proteolytic activities or perhaps a single proteolytic enzyme with broad amino acid specificity. We investigated the role of cathepsin D in the processing of βAPP since this enzyme has been widely proposed as a γ-secretase candidate. Treatment of a synthetic peptide that spans the γ-secretase site of βAPP with human cathepsin D resulted in the cleavage of this substrate at Ala42-Thr43. A sensitive liquid chromatography/mass spectrometry technique was also developed to further investigate the ability of cathepsin D to process longer recombinant βAPP substrates (156 and 100 amino acids of βAPP carboxyl terminus) in vitro. The precise cathepsin D cleavage sites within these recombinant βAPP substrates were identified using this technique. Both recombinant substrates were cleaved at the following sites: Leu49-Val50, Asp68-Ala69, Phe93-Phe94. No cleavages were observed at putative γ-secretase sites: Val40-Ile41 or Ala42-Thr43, suggesting that cathepsin D is not γ-secretase as defined by these βA4 termini. Under conditions where the βAPP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of βA4, Glu−3-Val−2 and Glu3-Phe4, were observed, indicating that cathepsin D cleavage of βAPP is influenced by the structural integrity of the substrate. Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as γ-secretase; however, the ability of this enzyme to efficiently cleave βAPP substrates at nonamyloidogenic sites within the molecule may reflect a role in βAPP catabolism.
All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members. This may be overcome by a HER ligand binding molecule that sequesters multiple EGF-like ligands, preventing ligand-dependent receptor activation. Toward this end, we have combined the HER-1/EGFR and HER-3 ligand binding domains, dimerized with fusion of an Fc fragment of human IgG1. This resulted in a mixture of HER-1/Fc homodimer (HFD100), HER-3/Fc homodimer (HFD300), and HER-1/Fc:HER-3/Fc heterodimer (RB200), also termed Hermodulins. The purified first-generation RB200 bound EGF and neuregulin 1 (NRG1)-beta1 ligands, determined by cross-linking and direct binding studies. The binding affinity for both was approximately 10 nmol/L by dissociation-enhanced lanthanide fluorescence immunoassay using europium (Eu)-labeled ligands. Competition studies with RB200 using Eu-EGF or Eu-NRG1-beta1 revealed that RB200 bound HER-1 ligands, including transforming growth factor-alpha and heparin-binding EGF, and HER-3 ligands NRG1-alpha and NRG1-beta3. RB200 inhibited EGF- and NRG1-beta1-stimulated tyrosine phosphorylation of HER family proteins, proliferation of a diverse range of tumor cells in monolayer cell growth assays, tumor cell proliferation as a single agent and in synergy with tyrosine kinase inhibitors, lysophosphatidic acid-stimulated cell proliferation, and tumor growth in two human tumor xenograft nude mouse models. Taken together, the data reveal that RB200 has the potential to sequester multiple HER ligands and interfere with signaling by HER-1, HER-2, and HER-3.
Abstract The eukaryotic serine protease, rat anionic trypsin, and various mutants created by site‐directed mutagenesis have been heterologously expressed in Escherichia coli . The bacterial alkaline phosphatase ( phoA ) promoter was used to control the expression of the enzymes in an induced or constitutive fashion. The DNA coding for the eukaryotic signal peptide of pretrypsinogen was replaced with DNA coding for the phoA signal peptide. The phoA signal peptide successfully directs the secretion of the mammalian trypsinogen to the periplasmic space of E. coli . Active trypsin was expressed in the periplasm of E. coli by deleting the DNA coding for the activation hexapeptide of the zymogen. The activity of trypsin in the periplasm suggests that the enzyme is correctly activated and has folded such that the 12 cysteine residues involved in the six disulfide bonds of rat anionic trypsin have paired correctly. A transcription terminator increased the level of expression by a factor of two. However, increasing the copy number of the plasmid decreased the levels of expression. Localization of the active enzyme in the periplasm allows rapid screening of modified trypsin activities and facilitates the purification of protein to homogeneity and subsequently to crystallinity.
Introduction: Medin, a 50 aa cleavage fragment of MFG-E8/ lactadherin appears to self assemble into fibrils and form the amyloid deposits seen in patients with aortic aneurysms. While the pathogenic nature of these aggregates is not fully understood, it appears medin may perturb smooth muscle cell function and thereby weaken the integrity of the aorta wall. One approach to treat aortic aneurysms may be to sequester medin and thereby block aggregation or remove the amyloid deposits from the aorta using a monoclonal antibody. Methods: Mice were immunized with a c-terminal peptide or full-length human medin, hybridomas cloned and antibodies screened for activity using ELISA, Western blot, Biacore and immunocytochemistry. Results: Initial ELISA studies revealed that antibodies raised against full-length 50 aa medin (e.g. 6B3) bind both medin peptide and MFG-E8, while antibodies raised against a c-terminal peptide (e.g. 18G1) appeared to be medin specific. Subsequent studies confirmed these observations and revealed that 18G1 recognized the c-terminal end of medin, a neo epitope created when medin is cleaved from MFG-E8. Data from Western blot also showed that both 6B3 and 18G1 were capable of binding both monomeric and oligomeric forms of medin. To assess the specificity for endogenous human medin, a series of immunohistochemical studies were conducted with thoracic aorta samples from aneurysm or Marfan syndrome patients. The results demonstrated that anti-medin antibodies were able to bind endogenous medin, although the intensity and pattern of staining appeared to be antibody specific. While 6B3 stained dense aggregated (Thioflavin S+) material or amyloid deposits with high affinity in and around the patient aneurysm, 18G1 showed little to no specific staining. In contrast, 18G1 widely stained structures in the tunica media, the region of the aorta that contains the elastin fibers and smooth muscle cells. Conclusions: These data demonstrated that anti-medin antibodies can detect endogenous medin peptide present in aneurysm and Marfan patient samples and showed that the antibody epitope can play an important role in determining the size (monomer, dimer, trimer, etc.) and aggregation state (oligomer, protofibril, aggregate) of medin detected.
Ser-neotrypsinogen and Val-neotrypsinogen are two-chain modifications of bovine trypsinogen produced on limited proteolysis with trypsin. Ser-neotrypsinogen has Lys131-Ser132 cleaved in the connecting peptide (the autolysis loop) linking the amino- and carboxyl-terminal domains. Val-neotrypsinogen has Arg105-Val106 cleaved which is located within the amino-terminal domain. The mixed disulfide derivative of Ser-neotrypsinogen was successfully refolded. A functional molecule was regenerated from the polypeptide fragments with the correct molecular weight of neotrypsinogen in an overall yield of 7%. Val-Neotrypsinogen could not be refolded. The first-order rate constants for the regeneration of Ser-neotrypsinogen were determined from the formation of active enzyme molecules as a function of time and from the regain of the correct molecular weight. Both kinetic values were the same indicating that refolding of the polypeptide chains first forms globular domain structures. The two domains then associate and the disulfide bonds between the domains and the correct geometry of the active site residues are formed last. The same kinetic results were also found in refolding Thr-neochymotrypsinogen (Duda, C. T., and Light, A. (1982) J. Biol. Chem. 257, 9866-9871) where peptide bond cleavage also occurred in the connecting peptide. These observations support the hypothesis that the pathway of folding of serine proteinases proceeds with the independent refolding of domains.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetection of intermediate species in the refolding of bovine trypsinogenAlbert Light and Jeffrey N. HigakiCite this: Biochemistry 1987, 26, 17, 5556–5564Publication Date (Print):August 25, 1987Publication History Published online1 May 2002Published inissue 25 August 1987https://pubs.acs.org/doi/10.1021/bi00391a051https://doi.org/10.1021/bi00391a051research-articleACS PublicationsRequest reuse permissionsArticle Views52Altmetric-Citations13LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
4076 Receptor tyrosine kinases such as members of the HER family are well-validated targets for cancer therapy. However, anti-cancer agents directed at only one HER family member often encounter endogenous or acquired resistance due to multiple HER co-activation or increased production of HER1 or HER3 ligands by tumor cells. To target multiple members of the HER family and block their function, we have developed a novel bi-specific HER1/HER3 heterodimeric ligand trap comprised of optimized HER1 and HER3 extracellular domains fused with the Fc portion of human IgG1. We investigated binding activities of a prototypic Hermodulin, RB200, using europium-labeled ligands to compare ligand binding affinities of RB200 with those of Hermodulin variants obtained via a high throughput rational mutagenesis process (optimized Hermodulins). Our data show that RB200 retains specific binding to various HER ligands, such as EGF, TGF-alpha, HB-EGF and neuregulin (NRG1-beta1) (Kd = 10 to 30 nM). Optimized Hermodulins have improved ligand binding affinities (Kd = 0.5 to 2 nM) for both HER1 and HER3 ligands with greater Bmax. We also developed a sensitive ELISA specific for Hermodulins, and studied the pharmacokinetics in rodent models in order to provide dosing strategies and interpret future studies evaluating the efficacy and tolerability of Hermodulins. RB200 was administered as a single iv or ip dose of 15-30 mg/kg in rats and mice, and plasma samples were analyzed via ELISA. RB200 exhibited high bioavailability, a low volume of distribution, and a prolonged terminal half-life consistent with expectations for Fc-fusion proteins and therapeutic monoclonal antibodies. Together these data suggest that Hermodulins act as bi-specific HER1/HER3 ligand traps and have desirable in vivo pharmacokinetic characteristics. These results support continued development of Hermodulins for cancer therapy.
AACR Centennial Conference: Translational Cancer Medicine-- July 20-23, 2008; Monterey, CA
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Resistance to single-targeted anti-HER agents has been attributed to multiple HER co-activation that occurs through receptor dimerization. We constructed and purified novel molecules comprising HER1/EGFR and HER3 ligand binding domains tethered to a human Ig Fc region. By design, these Hermodulins bind the majority of HER ligands and provide a therapeutic anti-cancer strategy similar to clinically-proven molecules developed for both TNF-alpha and VEGF-related diseases. RB200, a prototypic Hermodulin, was evaluated for activity against a spectrum of cell lines derived from human malignancies. Effects on monolayer cell growth in the presence of serum were measured. Dose-dependent inhibition of cell growth by RB200 was demonstrated in many cell lines. Synergistic growth inhibition in vitro was also demonstrated when RB200 was combined with small molecule tyrosine kinase inhibitors (TKIs) and conventional cytotoxic chemotherapies. Nude mice bearing established subcutaneous human NSCLC tumors, derived from the H1437 tumor cell line, were used to evaluate in vivo activity of RB200. Mice (N=10/group) were treated with RB200, PBS or cetuximab. Tumor sizes were measured via external calipers. These studies showed ~70% inhibition of tumor growth at 10 mg/kg vs. PBS control. Greater anti-tumor effects were observed when RB200 was combined with the TKIs. Optimized versions of Hermodulins with enhanced activity are being developed.
