Ca 2؉ -ATPase of sarcoplasmic reticulum is an ATP-powered Ca 2؉ pump but also a H ؉ pump in the opposite direction with no demonstrated functional role. Here, we report a 2.4-Å-resolution crystal structure of the Ca 2؉ -ATPase in the absence of Ca 2؉ stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca 2؉ binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by Ca 2؉ -release. They suggest that H ؉ countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca 2؉ -binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.2ϩ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a), an integral membrane protein consisting of 994 aa (1), transfers two Ca 2ϩ from the cytoplasm into the lumen of sarcoplasmic reticulum per ATP hydrolyzed and thereby establishes a Ͼ10 4 concentration gradient across the membrane (2). At the same time, Ca 2ϩ -ATPase pumps two or three H ϩ in the opposite direction (3-6) during the reaction cycle. According to the classical E1͞E2 theory (7-9), transmembrane ion-binding sites have high affinity for Ca 2ϩ and face the cytoplasm in E1, whereas they have low affinity and face the lumen of sarcoplasmic reticulum in E2. The opposite applies to H ϩ , which binds to the ATPase in E2 and dissociates in E1, presumably in exchange with Ca 2ϩ . As Na ϩ K ϩ -ATPase and gastric H ϩ K ϩ -ATPase countertransport K ϩ , instead of H ϩ , countertransport of monovalent cations may be a common feature of the P-type ATPase superfamily (2, 10), of which SERCA1a is the best-studied member (11,12). However, the sarcoplasmic reticulum membrane is leaky to monovalent cations including H ϩ (13). As has been pointed out before (14), the membrane potential and pH gradient must be minimized to achieve such a large concentration gradient of Ca 2ϩ . Therefore, the physiological role of H ϩ countertransport has been a puzzle. Although protonation of carboxyls in the Ca 2ϩ -binding site has been suggested from biochemical (15, 16) and mutagenesis studies (17, 18), crystal structures of SERCA1a in various states (19-24) did not clarify the role of countertransport or identify protonation sites.These questions, however, can be addressed computationally by calculating stabilization energy provided by H ϩ binding (25)(26)(27). Protonation probability of a particular residue is related directly to the free energy difference between protonated and unprotonated forms. Such calculations, therefore, should be able to identify residues likely to be protonated in crystal structures and were indeed successful (28) for a Ca 2ϩ -bound form [E1⅐2C...

Nakajo-Nishimura syndrome (NNS) is a disorder that segregates in an autosomal recessive fashion. Symptoms include periodic fever, skin rash, partial lipomuscular atrophy, and joint contracture. Here, we report a mutation in the human proteasome subunit beta type 8 gene (PSMB8) that encodes the immunoproteasome subunit β5i in patients with NNS. This G201V mutation disrupts the β-sheet structure, protrudes from the loop that interfaces with the β4 subunit, and is in close proximity to the catalytic threonine residue. The β5i mutant is not efficiently incorporated during immunoproteasome biogenesis, resulting in reduced proteasome activity and accumulation of ubiquitinated and oxidized proteins within cells expressing immunoproteasomes. As a result, the level of interleukin (IL)-6 and IFN-γ inducible protein (IP)-10 in patient sera is markedly increased. Nuclear phosphorylated p38 and the secretion of IL-6 are increased in patient cells both in vitro and in vivo, which may account for the inflammatory response and periodic fever observed in these patients. These results show that a mutation within a proteasome subunit is the direct cause of a human disease and suggest that decreased proteasome activity can cause inflammation.

Proteasomes are multisubunit proteases that play a critical role in maintaining cellular function through the selective degradation of ubiquitinated proteins. When 3 additional β subunits, expression of which is induced by IFN-γ, are substituted for their constitutively expressed counterparts, the structure is converted to an immunoproteasome. However, the underlying roles of immunoproteasomes in human diseases are poorly understood. Using exome analysis, we found a homozygous missense mutation (G197V) in immunoproteasome subunit, β type 8 (PSMB8), which encodes one of the β subunits induced by IFN-γ in patients from 2 consanguineous families. Patients bearing this mutation suffered from autoinflammatory responses that included recurrent fever and nodular erythema together with lipodystrophy. This mutation increased assembly intermediates of immunoproteasomes, resulting in decreased proteasome function and ubiquitin-coupled protein accumulation in the patient's tissues. In the patient's skin and B cells, IL-6 was highly expressed, and there was reduced expression of PSMB8. Downregulation of PSMB8 inhibited the differentiation of murine and human adipocytes in vitro, and injection of siRNA against Psmb8 in mouse skin reduced adipocyte tissue volume. These findings identify PSMB8 as an essential component and regulator not only of inflammation, but also of adipocyte differentiation, and indicate that immunoproteasomes have pleiotropic functions in maintaining the homeostasis of a variety of cell types.