Ca2+ signals triggered by bacterial pathogens and microdomains

Recent reports have highlighted the pivotal role of Ca²⁺ during host cell infection by bacterial pathogens. Here, we review how bacterial pore-forming toxins (PFTs) trigger global Ca²⁺ signals to regulate cell adhesion-, inflammatory- or death processes. We comment recent reports describing the role of bacterial effectors injected by a type III secretion system (T3SS) as well as host cell players in the formation of Ca²⁺ microdomains during Shigella invasion and Chlamydia extrusion of host cells. We discuss how modeling and comparison between bacterial-induced and physiological Ca²⁺ microdomains provides insight into the critical parameters shaping the duration of local Ca²⁺ responses.     

Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy

Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL‐1β is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R‐SNARE Sec22b to recruit cargo to the LC3‐II⁺ sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome–lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP‐23 and SNAP‐29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.     

Localization of a proton-translocating ATPase on sucrose gradients

Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing β-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin.     

Studies on 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase using chorismate mutase inhibitors

The proposed cyclic mechanism of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and the mechanism of chorismate mutase share certain structural and electronic similarities. In this report, we examine several inhibitors of chorismate mutase for their efficacy against KDO 8-P synthase.     

Identification of different species ofPenicillium causing deterioration of the Moroccan table grapes during storage

Penicillium glabrum, P. purpurescens, P. decumbens, P. expansum, P. chrysogenum, P. crustosum andP. aurantiogriseum are responsible for some of the alterations noticed on Moroccan table grapes cold stored. Contamination of these table grapes withPenicillium species occurs in the vineyard and inside the cooling station where other fruits which are often fungus-contaminated are also kept. All of thesePenicillium species were able to grow at 0°C apart fromP. glabrum andP. purpurescens. Consequently, refrigeration of grapes during long-term storage is not sufficient in itself in preserving their initial qualities.     

Autophagy regulation: RNF2 targets AMBRA1

The phosphatidylinositol 3-kinase complex I (PI3K complex I) is a crucial regulator of autophagy, which contains Beclin 1 (or ATG6), ATG14L, VPS34 (or the class III phosphatidylinositol 3-kinase and its adaptor VPS15) and AMBRA1, and controls autophagosome formation. In a paper recently published in Cell Research, Xia et al. report that during nutrient deprivation the ubiquitin E3 ligase RNF2 is recruited to the PI3K complex I, and ubiquitinates AMBRA1 to trigger its degradation and downregulate autophagy.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components¹. The ubiquitin-proteasome pathway is also critical during the process of autophagy. The formation of autophagosomes (double-membrane bound vacuoles that sequester cargo in bulk or in a selective manner before their delivery to the lysosomal compartment) depends on ubiquitination-like activity². The selective removal of cargo (e.g., protein aggregates, organelles) by autophagy is dependent in many cases on the ubiquitination of the cargo³. Recent studies also indicate that ubiquitination regulates the activity of autophagy proteins that comprise autophagosomes⁴.Autophagosome formation is dependent on evolutionarily conserved ATG (autophagy-related) proteins initially identified in yeast². These proteins function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via stages of initiation, elongation, and sealing. Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homolog of yeast ATG6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34² (Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P), is crucial to recruitment of ATG, which is required for the elongation and sealing of the autophagosomal membrane.Open in a separate windowFigure 1Role of RNF2 and WASH during autophagosome formation. Upon autophagy stimulation by nutrient deprivation, Beclin 1 forms a complex with VPS34 and its adaptor VPS15, ATG14L, and AMBRA1. In this complex, Beclin 1 is K63-polyubiquitylated (green circles) by the E3 ligase AMBRA1 to activate the production of PI3P by VPS34. The production of PI3P at the phagophore recruits WIPI proteins to trigger the ATG machinery to elongate and seal the membrane to form an autophagosome. After the induction of autophagy, WASH and RNF2 are recruited to downregulate the autophagy pathway by inhibiting the VPS34 activity. WASH negatively regulates autophagy through suppression of Beclin 1 K63-linked polyubiquitination whereas RNF2 is recruited to the complex via the Beclin 1 interactor WASH. RNF2 catalyzes K48-linked polyubiquitination (orange circles) of AMBRA1 to mediate its proteasomal degradation.Recently, Xia and colleagues⁵ demonstrate that RNF2 (also called Ring1B) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitination of AMBRA1. RNF2 is a member of the RING-domain ubiquitin E3 ligase family⁶. In a series of experiments involving two-hybrid screens with RNF2 as bait, Xia and colleagues⁵ showed that RNF2 interacts with AMBRA1 and that this interaction is enhanced upon autophagy stimulation in cells cultured in the absence of serum and amino acids. Deletion of RNF2 robustly stimulates autophagy in response to starvation whereas restoration of RNF2 in RNF2^−/− mouse embryo fibroblasts (MEFs) reduces autophagy. An RNF2 mutant with no E3 ligase activity does not impair autophagy in RNF2^−/− MEFs. The authors further showed that RNF2 downregulates autophagy by promoting the degradation of AMBRA1. RNF2 catalyzes the K48-linked polyubiquitination of AMBRA1 which mediates its proteasomal degradation. The crucial site for AMBRA1 K48-linked polyubiquitination is lysine 45, and a K45R AMBRA1 mutant is not sensitive to RNF2-mediated ubiquitination and is able to sustain VPS34 activity and autophagy. Beclin 1 exists in different complexes involved in different steps of the autophagic pathway². In the current study, the authors showed that RNF2 is associated with the PI3K complex 1 with Beclin 1 ATG14 and AMBRA1, a complex involved in early stage of autophagosome formation. Interestingly, in the absence of RNF2, the association of Beclin 1 with VPS34 in the PI3K complex 1 is enhanced.Recently, Fan''s group reported that AMBRA1-DDB1-CUL4A is the E3 ligase that mediates K63-linked ubiquitination of Beclin 1 to enhance its binding to VPS34 in response to starvation⁷. In the present study⁵, the group showed that the K63-linked ubiquitination of Beclin 1 is inhibited by RNF2. A screen identified WASH as an interactor of RNF2. WASH is part of a complex that promotes actin polymerization to facilitate endosomal protein sorting⁸ and has been recently shown to interact with Beclin 1 and to be associated with autophagosomal membrane⁷. This interaction impairs the AMBRA1-mediated K63 polyubiquitination of Beclin 1. WASH recruits RNF2 to AMBRA1 and the PI3K complex 1 in response to starvation. The absence of WASH abolishes the K48-linked polyubiquitination of AMBRA1. In addition, WASH overexpression partially impairs the interaction of AMBRA1 with Beclin1 to block its K63-linked polyubiquitination⁷. The study by Xia et al. reveals a novel layer of regulation: WASH recruits RNF2 to promote AMBRA1 degradation to impair Beclin 1 ubiquitination⁵. This work points to the complex role of ubiquitination in the regulation of the early stage of autophagy with a balance between activating K63-linked polyubiquitination of Beclin 1 and inhibitory K48-linked polyubiquitination of AMBRA1. These post-translational modifications depend on the activity of two E3 ligases, AMBRA1 and RNF2. This study also stresses the importance of AMBRA1 in stabilization of proteins engaged in the early stage of autophagosome formation via its E3 ligase activity with DDB1-CUL4A to enhance Beclin 1 association with VPS34⁷ and with TRAF6 to stabilize ULK1, which acts in a complex upstream of the PI3K complex 1 in autophagy⁹. Finally, the study by Xia et al. emphasizes that autophagy must be tightly regulated to avoid deleterious effects on cell homeostasis.The present study raises several questions regarding how and when WASH and RNF2 are recruited to downregulate autophagy in response to starvation. Recently it has been shown that WASH is a positive modulator of autophagosome biogenesis in mammalian cells through regulation of endosomal trafficking of ATG9A¹⁰. Altogether, these findings suggest that the role of WASH in autophagy is dependent on its subcellular localization and its partners in intracellular membranes.     

Radioimmunoassay of prostaglandins E1, E2, and F2α in unextracted plasma, serum and myocardium

A radioimmunoassay procedure for the determination of PGE₁, PGE₂, and PGF₂α is presented. The procedure involves the pre-precipitation of each prostaglandin specific antiserum with the precipitating antisera (ARGG), and the use of these antisera mixtures in assaying for PGE₁, PGE₂, and PGF₂α. Applicability of the methods to unextracted plasma, serum and myocardial homogenate has been demonstrated through tests of specificity, recovery, reproducability and parallelism. A mathematical correction for cross-reactivity between PGE₁ and PGE₂, and their opposing antisera is given. To demonstrate the utility of the methodology in differentiation of experimental variables, prostaglandin concentrations in unincubated serum, incubated serum, and the rate of prostaglandin production in serum of dogs are given.     

Ligand binding to inhibitory killer cell Ig-like receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.     

PCR-RFLP of ITS rDNA for the rapid identification of Penicillium subgenus Biverticillium species

RFLP of ITS rDNA is proposed as a useful tool for molecular identification of the most common species of biverticillate penicillia. 60 isolates were analysed representing 13 species and 21 unique sequences were produced. The combination of five restriction enzymes was successful in separating 12 species. However, the variety Penicillium purpurogenum var. rubrisclerotium remained indistinguishable from Penicillium funiculosum. P. funiculosum appeared as the most confused species, being mis-identified with Penicillium miniolutum and Penicillium pinophilum, which were originally part of the species, and with P. purpurogenum perhaps because of the common production of red pigment. Penicillium variabile was difficult to investigate as introns were found on half of the isolates. Penicillium piceum, Penicillium rugulosum, Penicillium loliense, Penicillium erythromellis and P. purpurogenum were homogeneous from molecular and morphological positions and corresponded to a well circumscribed taxon. Furthermore, intraspecific variability was evidenced within P. pinophilum and P. funiculosum. The ex-type isolate of P. funiculosum produced a unique pattern. The method is sensitive, rapid and inexpensive and can be used for isolate identification of the biverticillate species. It is recommended particularly when many isolates have to be authentificated prior to analysis for phylogenetic assessment or population genetics.