Selected BTB/POZ-kelch proteins bind ATP.

AbstractProteins with a bric-à-brac, tramtrack, broad-complex/Poxvirus zinc fingers (BTB/POZ) domain are implicated in a broad variety of biological processes, including DNA binding, regulation of gene transcription and organization of macromolecular structures. Kelch domain containing BTB/POZ proteins like Mayven and Keap1 display limited sequence similarity with the actin-fragmin kinase from Physarum, a protein kinase with a kelch domain. We show that mouse Keap1, a Caenorhabditis elegans protein that we named CKR, and human Mayven bind 5′-p-fluorosulfonyl-benzoyl-adenosine (FSBA), a covalently modifying ATP analogue. Binding with 2-azido-ATP or ATP-Sepharose is also demonstrated. In contrast to Mayven, FSBA binding by CKR and Keap1 was specifically inhibited by excess ATP. The ATP binding pocket is located in the N-terminal half of Keap1. Our findings indicate that several, but not all, BTB/POZ-kelch domain proteins possess an inconspicuous ATP binding cassette

Protein Profiling of Pleural Effusions to Identify Malignant Pleural Mesothelioma Using SELDI-TOF MS

Diagnosis of malignant pleural mesothelioma (MM) is limited. Novel proteomic technologies can be utilized to discover changes in expression of pleural proteins that might have diagnostic value. The objective of this study was to detect protein profiles that could be used to identify malignant pleural mesothelioma with surface enhanced laser desorption/ionization from time-of-flight (SELDI-TOF) mass spectrometry (MS). Pleural effusions were collected patients with confirmed mesothelioma (n = 41) and from patients with effusions due to other causes ([n = 48] cancerous and non-cancerous). Samples were fractionated using anion exchange chromatography and bound to different types of ProteinChip array surfaces. All samples were also subjected to other commercially available immunoassays (human epididymes protein 4 [HE4], osteopontin [OPN], soluble mesothelin-related proteins [SMRP], and the cytokeratin 19 fragment [CYFRA 21-1]). Peak intensity data obtained by SELDI-TOF were subjected to classification algorithms in order to identify potential classifier peaks. A protein peak at m/z 6614 was characterized as apolipoprotein (Apo) Cl. In this setting, the sensitivity and specificity of this potential biomarker was 76% and 69%, respectively. The area under the receiver operating characteristic curve (AUC) for Apo Cl was 0.755, thereby outperforming OPN, HE4, and CYFRA 21-1. SMRP performed best with an AUC of 0.860 with a sensitivity of 83% and specificity of 74%. Our study validates the use of SMRP as a diagnostic marker for pleural mesothelioma and furthermore suggests that Apo Cl levels could be used in the future to discriminate pleural mesothelioma from other causes of exudates