Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity

Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and βPix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or βPix, to membrane microdomains. Transient RNAi knockdown, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a βPix binding-deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or βPix in Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the Src homology 3 domain binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation of a catalytically active Nef-PAK2 complex.     

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria

Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2 = several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER–mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.     

In situ immobilized β-glucosidase fromAspergillus phoenicis QM 329

Summary Aspergillus phoenicis QM 329 was found to grow in the shape of beads in shake flasks and in an air-lift fermentor. Initial culture pH, pH profile during cultivation, carbon source, inoculum size and fermentor configuration influenced the retainment of the -glucosidase activty in the mycelium. Glucose and soluble starch produced beads with the highest activity and the best stability. Glucose-derived beads were more homogeneous in size and shape than the starch-derived beads. These beads kept their integrity for 10 d at 50° C. After 48 h hydrolysis of 50 g/l cellobiose 75% of the initial enzyme activity remained. The beads could be air-dried and alcohol-sterilized with only minor loss of activity. The size of the beads could be controlled by varying the size of the conidia inoculum. In an air-lift fermentor with a working volume of 1500 ml,900 ml beads with an average diameter of 2 mm (estimated to 37–45,000 beads) were produced in less than 3 d with glucose as carbon source. The beads held a -glucosidase activity of 0.140 IU/bead determined with the pNP assay.     

Proteome analysis of cultivated vascular smooth muscle cells from a CADASIL patient

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementing disease caused by mutations in the NOTCH3 gene, most which are missense mutations leading to an uneven number of cysteine residues in epidermal growth factor-like repeats in the extracellular domain of Notch3 receptor (N3ECD). CADASIL is characterized by degeneration of vascular smooth muscle cells (VSMC) and accumulation of N3ECD on the VSMCs of small and middle-sized arteries. Recent studies have demonstrated that impairment of Notch3 signaling is not the primary cause of the disease. In the present study we used proteomic analysis to characterize the protein expression pattern of a unique material of genetically genuine cultured human CADASIL VSMCs. We identified 11 differentially expressed proteins, which are involved in protein degradation and folding, contraction of VSMCs, and cellular stress. Our findings indicate that misfolding of Notch3 may cause endoplasmic reticulum stress and activation of unfolded protein response, leading to increased reactive oxygen species and inhibition of cell proliferation. In addition, upregulation of contractile proteins suggests an alteration in the signaling system of VSMC contraction. The accumulation of N3ECD on the cell surface possibly upregulates the angiotensin II regulatory feedback loop and thereby enhances the readiness of the cells to respond to angiotensin II stimulation.     

Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease

Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised. For the optimized substrate T-Abu-Q, with sequence Ac-Asp-Glu-Lys(TAMRA)-Glu-Glu-Abu-Psi(COO)Ala-Ser-Lys(QSY-7)-amide, the kinetic parameters with HCV NS3 protease are K(m)=30 microM, k(cat)=0.6s(-1), and k(cat)/K(m)=20,100s(-1)M(-1). We show that this substrate is suitable for inhibitor analysis and mechanistic studies so long as the substrate concentration is low enough (0.5 microM) to avoid complications from high inner filter effects. The substrate is especially useful with ultra-high-density screening formats, such as microarrayed compound screening technology, because there is less spectral interference from the compounds being tested than with more traditional (EDANS/DABCYL) FRET protease substrates. The merits of the new substrate, as well as potential applications of this FRET pair to other protease substrates, are discussed.     

Evidence for emerging parasites and pathogens influencing outbreaks of stress-related diseases like chalkbrood

In agriculture, honey bees play a critical role as commercial pollinators of crop monocultures which depend on insect pollination. Hence, the demise of honey bee colonies in Europe, USA, and Asia caused much concern and initiated many studies and research programmes aiming at elucidating the factors negatively affecting honey bee health and survival. Most of these studies look at individual factors related to colony losses. In contrast, we here present our data on the interaction of pathogens and parasites in honey bee colonies. We performed a longitudinal cohort study over 6 years by closely monitoring 220 honey bee colonies kept in 22 apiaries (ten randomly selected colonies per apiary). Observed winter colony losses varied between 4.8% and 22.4%; lost colonies were replaced to ensure a constant number of monitored colonies over the study period. Data on mite infestation levels, infection with viruses, Nosema apis and Nosema ceranae, and recorded outbreaks of chalkbrood were continuously collected. We now provide statistical evidence (i) that Varroa destructor infestation in summer is related to DWV infections in autumn, (ii) that V. destructor infestation in autumn is related to N. apis infection in the following spring, and most importantly (iii) that chalkbrood outbreaks in summer are related to N. ceranae infection in the preceding spring and to V. destructor infestation in the same season. These highly significant links between emerging parasites/pathogens and established pathogens need further experimental proof but they already illustrate the complexity of the host–pathogen-interactions in honey bee colonies.