Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.     

Autosomal-Dominant Striatal Degeneration Is Caused by a Mutation in the Phosphodiesterase 8B Gene

Autosomal-dominant striatal degeneration (ADSD) is an autosomal-dominant movement disorder affecting the striatal part of the basal ganglia. ADSD is characterized by bradykinesia, dysarthria, and muscle rigidity. These symptoms resemble idiopathic Parkinson disease, but tremor is not present. Using genetic linkage analysis, we have mapped the causative genetic defect to a 3.25 megabase candidate region on chromosome 5q13.3-q14.1. A maximum LOD score of 4.1 (Θ = 0) was obtained at marker D5S1962. Here we show that ADSD is caused by a complex frameshift mutation (c.94G>C+c.95delT) in the phosphodiesterase 8B (PDE8B) gene, which results in a loss of enzymatic phosphodiesterase activity. We found that PDE8B is highly expressed in the brain, especially in the putamen, which is affected by ADSD. PDE8B degrades cyclic AMP, a second messenger implied in dopamine signaling. Dopamine is one of the main neurotransmitters involved in movement control and is deficient in Parkinson disease. We believe that the functional analysis of PDE8B will help to further elucidate the pathomechanism of ADSD as well as contribute to a better understanding of movement disorders.     

Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

Electrical signals and their physiological significance in plants

Electrical excitability and signalling, frequently associated with rapid responses to environmental stimuli, are well known in some algae and higher plants. The presence of electrical signals, such as action potentials (AP), in both animal and plant cells suggested that plant cells, too, make use of ion channels to transmit information over long distances. In the light of rapid progress in plant biology during the past decade, the assumption that electrical signals do not only trigger rapid leaf movements in 'sensitive' plants such as Mimosa pudica or Dionaea muscipula, but also physiological processes in ordinary plants proved to be correct. Summarizing recent progress in the field of electrical signalling in plants, the present review will focus on the generation and propagation of various electrical signals, their ways of transmission within the plant body and various physiological effects.     

Enrichment strategies for siRNA-transfected cells in an untransfected background

Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.     

Calcium nutrition has a significant influence on wood formation in poplar

To test the effects of calcium on wood formation, Populus tremula x Populus tremuloides clones were supplied with Hoagland solution modified in its calcium contents. Energy-dispersive X-ray analysis (EDXA) revealed an increase in calcium in the phloem, the cambium and the xylem elongation zone with increasing Ca(2+) supply in the nutrient solution. Using light and electron microscopy, a strong impact was shown on the cambial and the elongation zones under calcium starvation. Using Fourier transform infrared (FTIR) spectroscopy on wood and bark cells formed under calcium starvation, we detected a reduction of some absorptions, such as carbonyl and methoxy groups from S-lignin. Also, a significant reduction in fiber length was detected with decreasing calcium supply in the nutrient solution. High-performance liquid chromatography (HPLC) analysis revealed a large increase in sugar concentrations in the leaves, but reduced concentrations in the bark under Ca(2+) deficiency. In conclusion, our results show a significant influence of calcium on the structure, chemistry and physiology of wood formation. Thus, efficient Ca(2+) supply has to be considered a decisive factor in wood formation.     

Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.     

Facilitation versus depression in cultured hippocampal neurons determined by targeting of Ca2+ channel Cavbeta4 versus Cavbeta2 subunits to synaptic terminals

Ca(2+) channel beta subunits determine the transport and physiological properties of high voltage-activated Ca(2+) channel complexes. Our analysis of the distribution of the Ca(v)beta subunit family members in hippocampal neurons correlates their synaptic distribution with their involvement in transmitter release. We find that exogenously expressed Ca(v)beta(4b) and Ca(v)beta(2a) subunits distribute in clusters and localize to synapses, whereas Ca(v)beta(1b) and Ca(v)beta(3) are homogenously distributed. According to their localization, Ca(v)beta(2a) and Ca(v)beta(4b) subunits modulate the synaptic plasticity of autaptic hippocampal neurons (i.e., Ca(v)beta(2a) induces depression, whereas Ca(v)beta(4b) induces paired-pulse facilitation [PPF] followed by synaptic depression during longer stimuli trains). The induction of PPF by Ca(v)beta(4b) correlates with a reduction in the release probability and cooperativity of the transmitter release. These results suggest that Ca(v)beta subunits determine the gating properties of the presynaptic Ca(2+) channels within the presynaptic terminal in a subunit-specific manner and may be involved in organization of the Ca(2+) channel relative to the release machinery.