Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex

TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.     

Degradation of nicastrin involves both proteasome and lysosome

The glycoprotein nicastrin (NCT) is an essential component of the gamma-secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph-1 and Pen-2. The gamma-secretase complex is not only involved in APP processing but also in the processing of an increasing number of other type I integral membrane proteins. As the largest subunit of the gamma-secretase complex, NCT plays a crucial role in its activation. Considerable information exists on the distribution, structure and function of NCT; however, little is known of its proteolysis. The present study is aimed at exploring the molecular mechanism of NCT degradation. We found that either proteasomal or lysosomal inhibition can significantly increase the levels of both endogenous and exogenous NCT in various cell lines, and the effect of these inhibitions on NCT was time- and dose-dependent. Immunofluorescent microscopic analysis revealed that NCT accumulates in the ER and Golgi apparatus after proteasomal inhibition, while lysosomal inhibition leads to the accumulation of NCT in the lysosomal apparatus. Co-immunoprecipitation can pull down both NCT and ubiquitin. Taken together, our results demonstrate that NCT degradation involves both the proteasome and the lysosome.     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

Brain MR Imaging and Proton MR Spectroscopy in Female Mice with Pyruvate Dehydrogenase Complex Deficiency

Pyruvate dehydrogenase complex (PDC) deficiency is an inborn metabolic disorder that causes neurological abnormalities. In this report, a murine model of PDC deficiency was analyzed using histology, magnetic resonance (MR) imaging and MR spectroscopy (MRS) and the results compared to PDC-deficient female patients. Histological analysis of brains from PDC-deficient mice revealed defects in neuronal cytoarchitecture in grey matter and reduced size of white matter structures. MR results were comparable to previously published clinical MR findings obtained from PDC-deficient female patients. Specifically, a 15.4% increase in relative lactate concentration, 64.4% loss of N-acetylaspartate concentration and a near complete loss of discernable glutamine plus glutamate concentration were observed in a PDC deficient mouse compared to wild-type control. Lower apparent diffusion coefficients (ADCs) were observed within the brain consistent with atrophy. These results demonstrate the usefulness of this murine model to systematically evaluate the beneficial effects of dietary and pharmacological interventions. Special issue dedicated to John P. Blass. Lioudmila Pliss and Richard Mazurchuk are two investigators who contributed equally.     

Branched Chain Keto-Acids Exert Biphasic Effects on α-Ketoglutarate-Stimulated Respiration in Intact Rat Liver Mitochondria

Pathophysiological concentrations of branched chain keto-acids (BCKAs), such as those that occur in maple syrup urine disease, inhibit oxygen consumption in liver homogenates and brain slices and the enzymatic activity of α-ketoglutarate- and pyruvate dehydrogenase complexes. Consistent with previous work, studies in isolated rat liver mitochondria indicate that three BCKAs, α-ketoisocaproate (KIC), α-keto-β-methylvalerate (KMV) and α-ketoisovalerate (KIV), preferentially inhibited State 3 respiration supported by α-ketoglutarate relative to succinate or glutamate/malate (KIC, >100-fold; KMV, >10-fold; KIV, >4-fold). KIC was also the most potent inhibitor (K_i,app 13 ± 2 μM). Surprisingly, sub-inhibitory concentrations of KIC and KMV can markedly stimulate State 3 respiration of mitochondria utilizing α-ketoglutarate and glutamate/malate, but not succinate. The data suggest that physiological concentrations of the BCKAs may modulate mitochondrial respiration. Special issue dedicated to John P. Blass.     

E1 component of pyruvate dehydrogenase complex does not regulate the expression of NADPH-ferredoxin reductase in Azotobacter vinelandii

In Azotobacter vinelandii, the E1 component of pyruvate dehydrogenase complex (PDHE1) is proposed to be a key regulatory protein in an oxidative stress management system that responds to superoxide. This proposal was tested by constructing an A. vinelandii mutant that had a disruption of aceE gene encoding PDHE1. This mutant exhibited wild-type exponential growth and a normal response to oxidative stress induced by paraquat. Electrophoretic mobility-shift assays revealed that a protein previously shown to bind to a paraquat-activatable DNA promoter was still present in the extract prepared from the mutant, implying that the protein cannot be PDHE1. These observations strongly contradict the previous claim that PDHE1 is a DNA-binding protein that is directly involved in the A. vinelandii oxidative stress-regulatory system.     

Diversity and distribution of Burkholderia cepacia complex in the rhizosphere of rice and maize

A survey of Burkholderia cepacia complex (Bcc) species was conducted in agricultural fields within Hangzhou, China. Out of the 251 bacterial isolates recovered on the selective media from the rhizosphere of rice and maize, 112 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the majority belong to B. cepacia, Burkholderia cenocepacia recA lineage IIIB, Burkholderia vietnamiensis and Burkholderia pyrrocinia. Burkholderia cenocepacia and B. vietnamiensis dominated the rhizosphere of maize and rice, respectively, indicating that species composition and abundance of Bcc may vary dramatically in different crop rhizospheres. In addition, one isolate (R456) formed a single discrete cluster within the phylogenetic analysis of the Bcc recA gene, and it may belong to a new genomovar.     

The gene expression profile of Bombyx mori silkgland

During prepupal stage, the genes expression in silkgland is considered as a model for gene expression and regulation of eukaryotes. Aiming to comprehensively interpret gene expression profile in the silkgland, we collected all currently available EST, complete cDNA and protein expression information and other gene expression testing data published before, and explored their roles in their function pathways level. With the analysis of interaction between the known proteins and putative bio-macromolecules partners in silico, we list our prediction results in the form of pathway classification and test some of their expressions by experiments.