An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.

Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication.

The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, nonreceptor tyrosine kinase (c-Src), as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pulldown assays revealed an interaction between NS5A and the Src homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact phosphotyrosine-binding competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A's interaction with the SH2 domain of c-Src.

Ammonia-weighted imaging by chemical exchange saturation transfer MRI at 3 T.

Hepatic encephalopathy (HE) is triggered by liver cirrhosis and is associated with an increased ammonia level within the brain tissue. The goal of this study was to investigate effects of ammonia on in vitro amide proton transfer (APT)-weighted chemical exchange saturation transfer (CEST) imaging in order to develop an ammonia-sensitive brain imaging method. APT-weighted CEST imaging was performed on phantom solutions including pure ammonia, bovine serum albumin (BSA), and tissue homogenate samples doped with various ammonia concentrations. All CEST data were assessed by magnetization transfer ratio asymmetry. In addition, optical methods were used to determine possible structural changes of the proteins in the BSA phantom. In vivo feasibility measurements were acquired in one healthy participant and two patients suffering from HE, a disease associated with increased brain ammonia levels. The CEST effect of pure ammonia showed a base-catalyzed behavior. At pH values greater than 5.6 no CEST effect was observed. The APT-weighted signal was significantly reduced for ammonia concentrations of 5mM or more at fixed pH values within the different protein phantom solutions. The optical methods revealed no protein aggregation or denaturation for ammonia concentrations less than 5mM. The in vivo measurements showed tissue specific and global reduction of the observed CEST signal in patients with HE, possibly linked to pathologically increased ammonia levels. APT-weighted CEST imaging is sensitive to changes in ammonia concentrations. Thus, it seems useful for the investigation of pathologies with altered tissue ammonia concentrations such as HE. However, the underlying mechanism needs to be explored in more detail in future in vitro and in vivo investigations.

The off-rate of monomers dissociating from amyloid-ß protofibrils.

The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid-ß peptide (Aß) has been implicated in the pathogenesis of Alzheimer disease. The determination of the kinetics of the individual association and dissociation reactions is hampered by the fact that forward and reverse reactions to/from different aggregation states occur simultaneously. Here, we report the kinetics of dissociation of Aß monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering specifically monomeric Aß was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. Aß protofibril dissociation into monomers follows exponential decay kinetics with a time constant of ∼2 h at 25 °C and an activation energy of 80 kJ/mol, values typical for high affinity biomolecular interactions. This study demonstrates the high kinetic stability of Aß protofibrils toward dissociation into monomers and supports the delineation of the Aß folding and assembly energy landscape.

Platelets contribute to amyloid-ß aggregation in cerebral vessels through integrin αIIbß3-induced outside-in signaling and clusterin release.

Cerebral amyloid angiopathy (CAA) is a vascular dysfunction disorder characterized by deposits of amyloid-ß (Aß) in the walls of cerebral vessels. CAA and Aß deposition in the brain parenchyma contribute to dementia and Alzheimer's disease (AD). We investigated the contribution of platelets, which accumulate at vascular Aß deposits, to CAA. We found that synthetic monomeric Aß40 bound through its RHDS (Arg-His-Asp-Ser) sequence to integrin αIIbß3, which is the receptor for the extracellular matrix protein fibrinogen, and stimulated the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin from platelets. Clusterin promoted the formation of fibrillar Aß aggregates, and ADP acted through its receptors P2Y1 and P2Y12 on platelets to enhance integrin αIIbß3 activation, further increasing the secretion of clusterin and Aß40 binding to platelets. Platelets from patients with Glanzmann's thrombasthenia, a bleeding disorder in which platelets have little or dysfunctional αIIbß3, indicated that the abundance of this integrin dictated Aß-induced clusterin release and platelet-induced Aß aggregation. The antiplatelet agent clopidogrel, which irreversibly inhibits P2Y12, inhibited Aß aggregation in platelet cultures; in transgenic AD model mice, this drug reduced the amount of clusterin in the circulation and the incidence of CAA. Our findings indicate that activated platelets directly contribute to CAA by promoting the formation of Aß aggregates and that Aß, in turn, activates platelets, creating a feed-forward loop. Thus, antiplatelet therapy may alleviate fibril formation in cerebral vessels of AD patients.