Assessment of the stability of an immunoadsorbent for the extracorporeal removal of Beta-2-microglobulin from blood.

BACKGROUND: Dialysis-related amyloidosis (DRA) is a devastating and costly condition that affects patients with end stage kidney disease. A key feature of DRA is the formation of amyloid fibrils, consisting primarily of beta2-microglobulin. Except for kidney transplantation, conventional kidney replacement therapies, which are based on nonspecific mechanisms, do not adequately address beta2-microglobulin removal. An antihuman beta2-microglobulin single-chain variable region antibody fragment (scFv) was developed to confer specificity to beta2-microglobulin removal during hemodialysis. METHODS: The scFv was immobilized onto agarose and characterized for beta2m binding capacity, thermal stability at 37 degrees C, regeneration capacity, storage conditions, and sterility. RESULTS: The beta2-microglobulin binding capacity was 1.3 mg/ml scFv gel. The immunoadsorbent is thermally stable, can be regenerated, stored short-term in 20% ethanol, lyophilized for long-term storage, and withstand process conditions similar to that of a patient's hemodialysis therapy. CONCLUSIONS: The results support further investigation of immobilized scFvs as a novel tool to remove beta2-microglobulin from blood.

Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding.

Monoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins.