Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies.

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six-transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd-numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge-pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt-bridge networks with a significant shift from inter- to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even-numbered helices can undergo a face rotation, whereas odd-numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor.

Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury.

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45ß (GADD45ß-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK's pathological activation. GADD45ß-I was engineered by optimizing the domain of the GADD45ß, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45ß-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen-glucose deprivation in vitro. Moreover, GADD45ß-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45ß-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45ß-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.

SAP97-mediated ADAM10 trafficking from Golgi outposts depends on PKC phosphorylation.

A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP.