The flavoprotein cryptochromes (CRYs) act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFbxl3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitylation and degradation. Yet, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound, and Fbxl3-Skp1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Strikingly, the F-box protein Fbxl3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved C-terminal tail and burying its PER-binding interface. This novel F-box protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitylation.
Background Hyperkalemia is a rare life‐threatening complication of red blood cell (RBC) transfusion. Stored RBCs leak intracellular potassium (K+) into the supernatant; irradiation potentiates the K+ leak. As the characteristics of patients and implicated RBCs have not been studied systematically, a multicenter study of transfusion‐associated hyperkalemia (TAH) in the pediatric population was conducted through the AABB Pediatric Transfusion Medicine Subsection. Study Design The medical records of patients <18 years old were retrospectively queried for hyperkalemia occurrence during or ≤12 h after the completion of RBC transfusion in a 1‐year period. Collected data included patient demographics, diagnosis, medical history, timing of hyperkalemia and transfusion, mortality, and RBC unit characteristics. Results/Findings A total of 3777 patients received 19,649 RBC units during the study period in four facilities. TAH was found in 35 patients (0.93%) in 37 occurrences. The patient median age and weight were 1.28 years and 9.80 kg, respectively. All patients had multiple serious comorbidities. There were 79 RBC units transfused in the TAH events; 62% were irradiated, and the median age of the units was 10 days. The median total RBC volume transfused ≤12 h before TAH was 24% of patient estimated total blood volume, and the median infusion rate (IR) was19.6 ml/kg/h. Mortality rate within 1 day after the TAH event was 20%. Conclusions The prevalence of TAH in children was low; however, the 1‐day mortality rate was 20%. Patients with multiple comorbidities may be at higher risk for TAH. The IR was higher for patients who had TAH than the IR threshold for safe transfusion.
Senataxin is a large 303 kDa protein linked to neuron survival, as recessive mutations cause Ataxia with Oculomotor Apraxia type 2 (AOA2), and dominant mutations cause amyotrophic lateral sclerosis type 4 (ALS4). Senataxin contains an amino-terminal protein-interaction domain and a carboxy-terminal DNA/RNA helicase domain. In this study, we focused upon the common ALS4 mutation, L389S, by performing yeast two-hybrid screens of a human brain expression library with control senataxin or L389S senataxin as bait. Interacting clones identified from the two screens were collated, and redundant hits and false positives subtracted to yield a set of 13 protein interactors. Among these hits, we discovered a highly specific and reproducible interaction of L389S senataxin with a peptide encoded by the antisense sequence of a brain-specific non-coding RNA, known as BCYRN1. We further found that L389S senataxin interacts with other proteins containing regions of conserved homology with the BCYRN1 reverse complement-encoded peptide, suggesting that such aberrant protein interactions may contribute to L389S ALS4 disease pathogenesis. As the yeast two-hybrid screen also demonstrated senataxin self-association, we confirmed senataxin dimerization via its amino-terminal binding domain and determined that the L389S mutation does not abrogate senataxin self-association. Finally, based upon detection of interactions between senataxin and ubiquitin–SUMO pathway modification enzymes, we examined senataxin for the presence of ubiquitin and SUMO monomers, and observed this post-translational modification. Our senataxin protein interaction study reveals a number of features of senataxin biology that shed light on senataxin normal function and likely on senataxin molecular pathology in ALS4.
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