ABSTRACT
Background@#The F-box and Leucine-rich Repeat Protein 5 (FBXL5), a member of the E3 ligases, is considered to be the central iron sensor in mammals. The cryo-EM structure of FBXL5 in complex with IRP2 and SKP1 was reported by Wang et.al. in 2020. Surprisingly, a 2Fe-2S cluster seemed to be responsible for the iron-sensing capability of FBXL5. @*Objectives@#To further explore the mechanism of its regulation, it is important to study the interaction of FBXL5 with other proteins under regulated conditions so we attempted to express FBXL5 in the hopes of studying its interaction with IRPs in vitro. @*Methodology@#Plasmids were constructed to express FBXL5 in Escherichia coli expression hosts. Purification of an MBP-fused FBXL5 and GST-fused FBXL5 were performed using affinity chromatography. Peptide Mass Fingerprinting, Circular Dichroism spectroscopy, and SEC-MALS were employed to analyze the purified MBP-FBXL5. GST-FBXL5 was also used in a pull-down assay with Iron Regulatory Protein 1 (IRP1). @*Results@#We are successful in expressing and partially purifying full-length FBXL5 using E. coli with the aid of a protein tag, the maltose binding protein (MBP) tag. However, cleavage of the protein tag resulted in decreased stability of FBXL5 as shown in SEC-MALS data. CD spectroscopy showed consistent secondary structure of FBXL5. A preliminary pull-down assay of GST-FBXL5 with IRP1 showed that IRP1 displayed interaction with the recombinant GST-FBXL5. @*Conclusion@#FBXL5, a 78-kDa mammalian protein was overexpressed in a prokaryotic expression system made stable by a fusion protein. The interaction of GST-FBXL5 with IRP1 also shows that it is possible to study their interaction in vitro.
Subject(s)
Leucine-Rich Repeat ProteinsABSTRACT
Recently, eosinophilic chronic rhinosinusitis (ECRS) has become a very common disease in Japan. ECRS differs from other types of sinusitis in terms of clinical features, such as high rate of recurrence, olfactory disorders, ineffective macrolide therapy, and severe paranasal sinus shadow on CT imaging. The diagnosis of ECRS requires a biopsy of nasal polyps. Therefore, we tried to predict ECRS by evaluating CT images using the Lund-Mackay CT Scoring system (LMS). This retrospective study involved 105 patients who had undergone surgical treatment (ESS) between July 2011 and November 2012. These patients were divided into three groups based on patterns of pathogenesis: ECRS, fungal sinusitis (FS), and chronic sinus infection (CSI). The pathological classification of nasal polyp was graded on a 3-point scale according to the degree of eosinophilic infiltration. Total LMS score for the ECRS was 9.4±0.6 (mean±SE), which was significantly higher than for the other LMS scores (FS: 4.6±1.0, CSI: 5.8±0.2). LMS scores for the maxillary sinus were high for all three patterns of pathogenesis (ECRS: 1.4± 0.1, FS: 1.7±0.2, CSI: 1.3±0.1). The scores for the frontal and sphenoid sinus forECRS were significantly higher than the scores for the FS and CSI (maxillary sinus, ECRS: 1.5±0.1, FS: 0.4±0.2, CSI: 0.6±0.1; sphenoid sinus, ECRS: 1.4±0.2, FS: 0.2±0.1, CSI: 0.6±0.3). We assessed for and found a positive correlation between the bilateral LMS score and eosinophilic infiltration (rs＝0.665). Therefore, ECRS can be predicted by LMS without biopsy and LMS helps determine the management of chronic rhinosinusitis.