Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells.

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.

Newly observed spontaneous activation of ethephon as a butyrylcholinesterase inhibitor.

The plant growth regulator ethephon (2-chloroethylphosphonic acid) inhibits human butyrylcholinesterase (BChE) by making a covalent adduct on the active site serine 198. Our goal was to extend earlier studies on ethephon inhibition. Addition of freshly prepared ethephon to BChE in buffered medium, at pH 7.0 and 22 °C, resulted in no inhibition initially. However, inhibition developed progressively over 60 min of incubation. Preincubation of ethephon in pH 7-9 buffers increased its initial inhibitory potency. These observations indicated that ethephon itself was not the inhibitor. About 3% of the initial ethephon could be trapped as a BChE adduct. Mass spectral analysis of the active site peptide from inhibited BChE showed that the inhibitor added a mass of 108 Da to the active site serine on peptide FGES198AGAAS. This result rules out a previous hypothesis that ethephon adds HPO3 to BChE (added mass of 80 Da). To accommodate these observations, we propose that in aqueous media at neutral to slightly alkaline pH about 3% of the ethephon is converted (t1/2 = 9.9 h at pH 7.0) into a cyclic oxaphosphetane which is the actual BChE inhibitor forming the 2-hydroxyethylphosphonate adduct on BChE at Ser198 while about 97% of the ethephon breaks down to ethylene (t1/2 = 11-48 h at pH 7.0) which is responsible for plant growth regulation.

Cresyl saligenin phosphate, an organophosphorus toxicant, makes covalent adducts with histidine, lysine, and tyrosine residues of human serum albumin.

CBDP [2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide] is a toxic organophosphorus compound. It is generated in vivo from tri-ortho-cresyl phosphate (TOCP), a component of jet engine oil and hydraulic fluids. Exposure to TOCP was proven to occur on board aircraft by finding CBDP-derived phospho-butyrylcholinesterase in the blood of passengers. Adducts on BChE, however, do not explain the toxicity of CBDP. Critical target proteins of CBDP are yet to be identified. Our goal was to facilitate the search for the critical targets of CBDP by determining the range of amino acid residues capable of reacting with CBDP and characterizing the types of adducts formed. We used human albumin as a model protein. Mass spectral analysis of the tryptic digest of CBDP-treated human albumin revealed adducts on His-67, His-146, His-242, His-247, His-338, Tyr-138, Tyr-140, Lys-199, Lys-351, Lys-414, Lys-432, and Lys-525. Adducts formed on tyrosine residues were different from those formed on histidines and lysines. Tyrosines were organophosphorylated by CBDP, while histidine and lysine residues were alkylated. This is the first report of an organophosphorus compound with both phosphorylating and alkylating properties. The o-hydroxybenzyl adduct on histidine is novel. The ability of CBDP to form stable adducts on histidine, tyrosine, and lysine allows one to consider new mechanisms of toxicity from TOCP exposure.

Cbl interacts with multiple E2s in vitro and in cells.

Many receptor tyrosine kinases (RTKs, such as EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. RF E3s, such as the Cbl proteins, interact with a ubiquitin-conjugating enzyme (E2) to confer specificity to the ubiquitination process and direct the transfer of ubiquitin from the E2 to one or more lysines on the target proteins. Using in vitro E3 assays and yeast two-hybrid screens, we found that Ube2d, Ube2e families, Ube2n/2v1, and Ube2w catalyze autoubiquitination of the Cbl protein and Ube2d2, Ube2e1, and Ube 2n/2v1 catalyze Cbl-mediated substrate ubiquitination of the EGFR and SYK. Phosphorylation of the Cbl protein by by Src resulted in increased E3 activity compared to unphosphorylated cbl or Cbl containing a phosphomimetic Y371E mutation. Ubiquitin chain formation depended on the E2 tested with Cbl with Ube2d2 forming both K48 and K63 linked chains, Ube2n/2v1 forming only K63 linked chains, and Ube2w inducing monoubiquitination. In cells, the Ube2d family, Ube2e family, and Ube2n/2v1 contributed to EGFR ubiquitination. Our data suggest that multiple E2s can interact with Cbl and modulate its E3 activity in vitro and in cells.

Molecular pathways: cbl proteins in tumorigenesis and antitumor immunity-opportunities for cancer treatment.

The Cbl proteins are a family of ubiquitin ligases (E3s) that regulate signaling through many tyrosine kinase-dependent pathways. A predominant function is to negatively regulate receptor tyrosine kinase (RTK) signaling by ubiquitination of active RTKs, targeting them for trafficking to the lysosome for degradation. Also, Cbl-mediated ubiquitination can regulate signaling protein function by altered cellular localization of proteins without degradation. In addition to their role as E3s, Cbl proteins play a positive role in signaling by acting as adaptor proteins that can recruit signaling molecules to the active RTKs. Cbl-b, a second family member, negatively regulates the costimulatory pathway of CD8 T cells and also negatively regulates natural killer cell function. The different functions of Cbl proteins and their roles both in the development of cancer and the regulation of immune responses provide multiple therapeutic opportunities. Mutations in Cbl that inactivate the negative E3 function while maintaining the positive adaptor function have been described in approximately 5% of myeloid neoplasms. An improved understanding of how the signaling pathways [e.g., Fms-like tyrosine kinase 3 (Flt3), PI3K, and signal transducer and activator of transcription (Stat)] are dysregulated by these mutations in Cbl has helped to identify potential targets for therapy of myeloid neoplasms. Conversely, the loss of Cbl-b leads to increased adaptive and innate antitumor immunity, suggesting that inhibiting Cbl-b may be a means to increase antitumor immunity across a wide variety of tumors. Thus, targeting the pathways regulated by Cbl proteins may provide attractive opportunities for treating cancer.

Cresyl saligenin phosphate makes multiple adducts on free histidine, but does not form an adduct on histidine 438 of human butyrylcholinesterase.

Cresyl saligenin phosphate (CBDP) is a suspected causative agent of "aerotoxic syndrome", affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106 amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free L-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins.

Reaction of human albumin with aspirin in vitro: mass spectrometric identification of acetylated lysines 199, 402, 519, and 545.

The aspirin esterase activity of human plasma is due to butyrylcholinesterase and albumin. Our goal was to identify the amino acid residues involved in the aspirin esterase activity of albumin. Fatty acid-free human albumin and human plasma were treated with aspirin for 5 min-24 h. Acetylated residues were identified by LC/MS/MS and MALDI-TOF/TOF mass spectrometry of tryptic peptides. Treatment with 0.3 mM aspirin resulted in acetylation of Lys-199, Lys-402, Lys-519, and Lys-545. Treatment with 20 mM aspirin resulted in acetylation of 26 lysines. There was no acetylation of Tyr-411, under any conditions. Acetylated lysine was stable for at least 21 days at pH 7.4, 37 degrees C. Albumin acetylated by aspirin had reduced esterase activity with beta-naphthyl acetate as shown on gels stained for esterase activity. It was concluded that the aspirin esterase activity of albumin is a pseudo-esterase activity in which aspirin stably acetylates lysines and releases salicylate.