The sequence, structural, and functional diversity within a protein family and implications for specificity and safety: The case for ETX_MTX2 insecticidal proteins.

The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common ß-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although ß-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the "relatedness"). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).

Herstatin, an autoinhibitor of the epidermal growth factor receptor family, blocks the intracranial growth of glioblastoma.

PURPOSE: Herstatin, an autoinhibitor of the epidermal growth factor (EGF) receptor family, was evaluated for efficacy against human glioblastoma in vitro and in vivo in a rat intracranial model. EXPERIMENTAL DESIGN: Glioblastoma controlled by EGF receptor (EGFR; U87MG) or by the truncated mutant, DeltaEGFR (U87MG/Delta), were transfected with Herstatin and evaluated for in vitro and in vivo growth in nude rat brain. Cells treated with purified Herstatin in vitro were evaluated for growth and signal transduction. RESULTS: Herstatin expression prevented tumor formation by U87MG and purified Herstatin inhibited their growth in vitro in a dose-responsive fashion, whereas in vivo and in vitro growth of U87MG/Delta was resistant to Herstatin. Inhibition of U87MG growth correlated with suppressed EGF activation of EGFR and of Akt but not mitogen-activated protein kinase signaling pathways, whereas DeltaEGFR activity and intracellular signaling in U87MG/Delta were unaffected by Herstatin treatment. CONCLUSIONS: Herstatin may have utility against glioblastoma driven by the EGFR but not the mutant DeltaEGFR. Blockade of Akt but not the mitogen-activated protein kinase signaling cascade appears to be critical for suppression of intracranial tumor growth.

Receptor binding specificities of Herstatin and its intron 8-encoded domain.

Retention of intron 8 in alternative HER-2 mRNA generates an inhibitory secreted ligand, Herstatin, with a novel receptor-binding domain (RBD) encoded by the intron. This study examines binding interactions with several receptors and investigates sequence variations in intron 8. The RBD, expressed as a peptide, binds at nM concentrations to HER-2, the EGFR, DeltaEGFR, HER-4 and to the IGF-1 receptor, but not to HER-3 nor to the FGF-3 receptor, whereas a rare mutation in the RBD (Arg to Ile) eliminates receptor binding. The full-length Herstatin binds with 3-4-fold higher affinity than its RBD, but with approximately 10-fold lower affinity to the IGF-IR. Sequence conservation in rhesus monkey but not in rat suggests that intron 8 recently diverged as a receptor-binding module critical for the function of Herstatin.

Polymorphisms of GSTT1 and related genes in head and neck cancer risk.

BACKGROUND: Glutathione S-transferase T1 detoxifies some environmental carcinogens while activating others and is deleted in 15% to 38% of humans. We sought to determine whether GSTT1 genotype and genotypes of several related genes are associated with risk of squamous cell carcinoma of the head and neck (HNSCC). METHODS: Somatic genotypes for GSTT1, GSTM1, GSTP1, and CYP1A1 were determined in 283 individuals with HNSCC and 208 population-based controls. RESULTS: The OR for presence of GSTT1 was 1.6 (CI, 1.1-2.5, p = 0.03). HNSCC risk was not associated with GSTM1 null genotype, the presence of the GSTP1 Val/Val genotype, or the Val/Val homozygous genotype for CYP1A1. Stratified analysis revealed disparate ORs for women (OR, 3.0; CI, 1.5-6.3) and men (OR, 1.2; CI, 0.7-2.1) for the presence of GSTT1. CONCLUSIONS: In this population, the presence of GSTT1 gene was associated with a significant increase in the risk of HNSCC. This association was particularly robust in women.

NH(2)-terminal truncated HER-2 protein but not full-length receptor is associated with nodal metastasis in human breast cancer.

BACKGROUND: The full-length receptor p185HER-2 undergoes a metalloprotease-dependent cleavage producing a membrane-associated fragment (p95HER-2) in cultured breast cancer cells. P95HER-2 has potentially enhanced signaling activity, but its expression and role in human breast cancer is poorly characterized. PURPOSE: The purpose of this project was to characterize the expression of p95HER-2 in primary breast cancers and nodal metastasis, and to study association with clinicopathological factors. EXPERIMENTAL DESIGN: P95HER-2 and p185HER-2 were examined in 337 primary breast tumors and 81 metastatic lymph nodes by Western blot analysis, and tested for associations with other clinicopathological factors. RESULTS: P95HER-2 was present in 20.9% of primary tumors from node-negative patients, in 29.1% from patients with one to three metastatic nodes, and in 36.7% from patients with four or more metastatic nodes (P = 0.027). Whereas p185HER-2 overexpression was unrelated to nodal disease (P = 0.63), the odds of lymph node metastasis were enhanced 2.9-fold by the presence of p95HER-2 (48.8% of node-negative versus 73.5% of node-positive patients; P = 0.03; odds ratio = 2.9). P95HER-2 was more frequent in metastatic lymph nodes than in primary tumors (45.7% versus 26.7%; P = 0.0009), whereas p185HER-2 overexpression was similar in both (22.3% versus 23.5%; P = 0.933). P95HER-2 did not significantly correlate with patient age, tumor size, stage, histotype, or hormone receptor status. CONCLUSIONS: P95HER-2 in primary tumors was related to extent of lymph node involvement and was enhanced in nodal tissue suggesting an important role as a marker or cause in breast cancer metastasis. Examination of the prognostic value of p95HER-2 in breast cancer and its coexpression with metalloprotease activity seem warranted.