Comparative Structural Analysis of Human DEAD-Box RNA Helicases

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members

Molecular Determinants of Survival Motor Neuron (SMN) Protein Cleavage by the Calcium-Activated Protease, Calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN

Mitochondrial dysfunction and biogenesis: do ICU patients die from mitochondrial failure?

Mitochondrial functions include production of energy, activation of programmed cell death, and a number of cell specific tasks, e.g., cell signaling, control of Ca2+ metabolism, and synthesis of a number of important biomolecules. As proper mitochondrial function is critical for normal performance and survival of cells, mitochondrial dysfunction often leads to pathological conditions resulting in various human diseases. Recently mitochondrial dysfunction has been linked to multiple organ failure (MOF) often leading to the death of critical care patients. However, there are two main reasons why this insight did not generate an adequate resonance in clinical settings. First, most data regarding mitochondrial dysfunction in organs susceptible to failure in critical care diseases (liver, kidney, heart, lung, intestine, brain) were collected using animal models. Second, there is no clear therapeutic strategy how acquired mitochondrial dysfunction can be improved. Only the benefit of such therapies will confirm the critical role of mitochondrial dysfunction in clinical settings. Here we summarized data on mitochondrial dysfunction obtained in diverse experimental systems, which are related to conditions seen in intensive care unit (ICU) patients. Particular attention is given to mechanisms that cause cell death and organ dysfunction and to prospective therapeutic strategies, directed to recover mitochondrial function. Collectively the data discussed in this review suggest that appropriate diagnosis and specific treatment of mitochondrial dysfunction in ICU patients may significantly improve the clinical outcome