Calpains (calcium-dependent cytoplasmic cysteine proteinases) are implicated in processes such as cytoskeleton remodeling and signal transduction. The 2.3-Å crystal structure of full-length heterodimeric [80-kDa (dI-dIV) ؉ 30-kDa (dV؉dVI)] human m-calpain crystallized in the absence of calcium reveals an oval disc-like shape, with the papain-like catalytic domain dII and the two calmodulin-like domains dIV؉dVI occupying opposite poles, and the tumor necrosis factor ␣-like ␤-sandwich domain dIII and the N-terminal segments dI؉dV located between. Compared with papain, the two subdomains dIIa؉dIIb of the catalytic unit are rotated against one another by 50°, disrupting the active site and the substrate binding site, explaining the inactivity of calpains in the absence of calcium. Calcium binding to an extremely negatively charged loop of domain dIII (an electrostatic switch) could release the adjacent barrel-like subdomain dIIb to move toward the helical subdomain dIIa, allowing formation of a functional catalytic center. This switch loop could also mediate membrane binding, thereby explaining calpains' strongly reduced calcium requirements in vivo. The activity status at the catalytic center might be further modulated by calcium binding to the calmodulin domains via the Nterminal linkers.T he calpains (EC 3.4.22.17; Clan CA, family C02) are a family of calcium-dependent cytosolic cysteine proteinases. They seem to catalyze limited proteolysis of proteins involved in cytoskeletal remodeling and signal transduction but are also implicated in other physiological and pathophysiological processes, such as cell cycle regulation, apoptosis, muscular dystrophies, cataractogenesis, and Alzheimer's or Parkinson's diseases (1-5). In mammals, the calpain family comprises several ''tissuespecific'' isoforms (n-calpains) besides two ''ubiquitous'' isoenzymes (-and m-calpains). In lower organisms such as insects, nematodes, fungi, and yeast, a number of ''atypical'' calpain homologues have been found.The ubiquitous -and m-calpains (calpains I and II), by far the best characterized calpains, are heterodimers comprising distinct but quite homologous 80-kDa ''large'' L-subunits and a common 30-kDa ''small'' S-subunit. On the basis of amino acid homologies, the L-and S-subunits have been described as consisting of four domains, dI to dIV, and of two domains, dV and dVI, respectively, with domain dII somewhat resembling papain and the calmodulin-like domains dIV and dVI containing EF-hands (6, 7). On exposure to calcium at concentrations of 5-50 M (-calpain) and 200-1,000 M (m-calpain), both calpains are activated and partially autolyzed. In vivo, both calpains seem to be active at physiological calcium concentrations of 100-300 nM, however, suggesting that other factors such as phospholipids might play a role in activation in addition.The crystal structures of rat and porcine domain dVI in the absence and presence of calcium have been determined (8, 9). For a full understanding of the activation mechanism and the functioning of calp...

Reactive oxygen species (ROS) are generated by virally-infected cells however the physiological significance of ROS generated under these conditions is unclear. Here we show that inflammation and cell death induced by exposure of mice or cells to sources of ROS is not altered in the absence of canonical ROS-sensing pathways or known cell death pathways. ROS-induced cell death signaling involves interaction between the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5−/− mice show exacerbated lung inflammation and proinflammatory cytokines in an ozone exposure model. Similarly, challenge with influenza A virus leads to increased virus infiltration, lymphocytic bronchiolitis and reduced survival of Pgam5−/− mice. This pathway, which we term ‘oxeiptosis’, is a ROS-sensitive, caspase independent, non-inflammatory cell death pathway and is important to protect against inflammation induced by ROS or ROS-generating agents such as viral pathogens.

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Auditory brainstem neurons that are involved in the precise analysis of the temporal pattern of sounds have ionic currents activated near the resting potential to shorten membrane time constants. One of these currents is the hyperpolarization-activated current (Ih). Molecular cloning of the channels underlying Ih revealed four different isoforms (HCN1-4). HCN1 and HCN2, which are widely distributed in the brain, differ in their activation kinetics, voltage dependence and sensitivity to cAMP. We determined the distribution of the HCN1 and HCN2 isoform in the auditory brainstem and midbrain of young rats (P20-30), using standard immunohistochemical techniques. HCN1 antibodies gave rise to punctate staining on the somatic and dendritic membrane. Strong HCN1 staining was present on octopus and bushy cells of the ventral cochlear nucleus, principal neurons of the lateral and medial superior olive, and neurons of the ventral nucleus of the lateral lemniscus. No HCN1 staining was observed in the dorsal cochlear nucleus and the medial nucleus of the trapezoid body (MNTB). In contrast, HCN2 staining was strongest in the MNTB and the dorsal nucleus of the lateral lemniscus. Strong HCN2 antibody labelling was also observed in bushy cells of the ventral cochlear nucleus. In the central nucleus of the inferior colliculus only a subpopulation of neurons showed HCN1 or HCN2 immunolabelling. This differential distribution of HCN1 and HCN2 channels is in agreement with the physiologically observed Ih currents in corresponding neuronal populations and might represent the basis for functional heterogeneity and diverse sensitivity to neuromodulators.