Miracle-dz/UniFAR-FTUs huggingface dataset:
Protein phosphatase 2A PR130/B”α:1 subunit binds to the SH2 domain‐containing inositol polyphosphate 5‐phosphatase 2 and prevents epidermal growth factor (EGF)‐induced EGF receptor degradation sustaining EGF‐mediated signaling

To elucidate novel cell biological functions of specific protein phosphatase 2A (PP2A) holoenzymes, we identified and biochemically characterized a complex between the Src homology 2 (SH2) domain‐containing inositol polyphosphate 5‐phosphatase 2 (SHIP2) and a PP2A holoenzyme comprising PR130/B″α1 as a regulatory subunit (PP2AT130)in several mammalian cell lines. PR130/B″α1 and SHIP2 partially colocalize in untreated HeLa cells, and both translocate to the cell membrane on epidermal growth factor (EGF) stimulation. Concomitantly, a transient EGF‐dependent interaction of PR130/B″α1 with the EGF receptor (EGFR) was observed, whereas the SHIP2‐PR130 interaction remained constitutive. As previously reported for SHIP2, RNA interference‐mediated knockdown of PR130 in COS‐7 cells resulted in increased EGF‐induced proteasome‐dependent EGFR degradation, and an increased interaction of EGFR with the E3 ligase c‐Cbl. In concordance with faster EGFR clearance or desensitization, intrinsic EGFR kinase activity (phospho‐Tyr‐1068) and downstream protein kinase B and extracellular signal‐regulated kinase/mitogen‐activated protein kinase pathways were more rapidly inactivated in PR130‐knockdown cells. Notably, these effects could be rescued by reintroduction of RNA interference‐resistant Myc‐PR130, excluding any off‐target effect. These data highlight a novel biological role of the PP2AT130 holoenzyme in EGF signaling through interaction with EGFR and the phosphatidylinositol (3,4,5)‐trisphosphate 5‐phosphatase SHIP2. This interaction may be of clinical relevance as dysfunction of EGF‐mediated signaling has been linked to various human cancers. —Zwaenepoel, K. , Goris, J., Erneux, C. , Parker, P.J., Janssens, V. Protein phosphatase 2A PR130/B″α1 subunit binds to the SH2 domaincontaining inositol polyphosphate 5‐phosphatase 2 and prevents epidermal growth factor (EGF)‐induced EGF receptor degradation sustaining EGF‐mediated signaling. FASEB J. 24, 538–547 (2010). www.fasebj. org
Questions about gonococcal pilus phase‐ and antigenic variation

Pathogenic organisms inhabit one of several defined locations within a host where temperature, pH, and nutrients are relatively constant. While the microorganism must adapt to different environments within the host, the host immune system is the most formidable predator that can limit the growth of a pathogen. Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of gonorrhoea, and has evolved several systems for varying the antigenicity of different surface antigens, presumably to help evade the effects of the human immune system. The On/Off/On phase variation of surface structure expression also alters the antigenic characteristics of the bacterial cell surface. Antigenic variation of the major subunit of the pilus, pilin, occurs by unidirectional, homologous recombination between a silent locus and the expression locus. The silent loci lie from 1 to 900 kb from the expression locus in the chromosome yet all can donate their sequences to the expression locus. The genetic composition of the pilin loci of two Gc strains has been elucidated, and the types of changes that lead to altered forms of the pilus have been extensively characterized. However, little is known about the precise molecular mechanisms used to allow high‐frequency, non‐reciprocal, chromosomal recombination between pilin loci or about what regulates the process of maintaining chromosome fidelity.
Preferential localization of the endocytic internalization machinery to hyphal tips underlies polarization of the actin cytoskeleton in Aspergillus nidulans

AbpA, SlaB and AmpA, three demonstrated components of the endocytic internalization machinery, are strongly polarized in Aspergillus nidulans hyphae, forming a ring that embraces the hyphal tip, leaving an area of exclusion at the apex. AbpA, a prototypic endocytic internalization marker, localizes to highly motile and transient (average half life, 24 ± 5 s) peripheral punctate structures overlapping with actin patches, which also predominate in the tip. SlaB also localizes to peripheral patches, but these are markedly more abundant and cortical than those of AbpA. In contrast to its polarized distribution in hyphae, endocytic patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarize as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along the hyphae, the apical predominance and the spatial organization of actin patches and of the above endocytic machinery proteins as a slightly subapical ring strongly suggests that tight spatial coupling of apical secretion and subapical compensatory endocytosis underlies hyphal growth. In agreement, the phenotype of a null slaB allele indicates that endocytosis is essential.
Differential effects of the dynein-regulatory factor Lissencephaly-1 on processive dynein-dynactin motility

Cytoplasmic dynein is the primary minus-end–directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins. Lissencephaly-1 (LIS1) is a highly conserved dynein-regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein-regulatory factor, releases dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here, we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we found that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bound up to two LIS1 dimers, and mutational analysis suggested that LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhanced DDB velocity in a concentration-dependent manner, in contrast to observations of the effect of LIS1 on the motility of isolated dynein. Thus, LIS1 exerts concentration-dependent effects on dynein motility and can synergize with dynactin to enhance processive dynein movement. Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly.
Characterization of ExsA and of ExsA‐dependent promoters required for expression of the Pseudomonas aeruginosa type III secretion system

Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is activated by ExsA, a member of the AraC/XylS family of transcriptional regulators. In the present study we examine the DNA‐binding properties of ExsA. ExsA was purified as a histidine‐tagged fusion protein (ExsAHis) and found to be monomeric in solution. ExsAHis specifically bound T3SS promoters with high affinity as determined by electrophoretic mobility shift assays (EMSA). For each promoter tested two distinct ExsA–DNA complexes were detected. Biochemical analyses indicate that the higher‐mobility complex consists of a single ExsAHis molecule bound to DNA while the lower‐mobility complex results from the binding of two ExsAHis molecules. DNase I protection assays demonstrate that the ExsAHis binding site overlaps the −35 RNA polymerase binding site and extends upstream an additional ∼34 bp. An alignment of all 10 ExsA‐dependent promoters revealed a number of highly conserved nucleotides within the footprinted region. We find that most of the highly conserved nucleotides are required for transcription in vivo; EMSA‐binding assays confirm that several of these nucleotides are essential determinants of ExsAHis binding. The combined data support a model in which two ExsAHis molecules bind adjacent sites on the promoter to activate T3SS gene transcription.
Polarized growth in fungi – interplay between the cytoskeleton, positional markers and membrane domains

One kind of the most extremely polarized cells in nature are the indefinitely growing hyphae of filamentous fungi. A continuous flow of secretion vesicles from the hyphal cell body to the growing hyphal tip is essential for cell wall and membrane extension. Because microtubules (MT) and actin, together with their corresponding motor proteins, are involved in the process, the arrangement of the cytoskeleton is a crucial step to establish and maintain polarity. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, actin‐mediated vesicle transportation is sufficient for polar cell extension, but in S. pombe, MTs are in addition required for the establishment of polarity. The MT cytoskeleton delivers the so‐called cell‐end marker proteins to the cell pole, which in turn polarize the actin cytoskeleton. Latest results suggest that this scenario may principally be conserved from S. pombe to filamentous fungi. In addition, in filamentous fungi, MTs could provide the tracks for long‐distance vesicle movement. In this review, we will compare the interaction of the MT and the actin cytoskeleton and their relation to the cortex between yeasts and filamentous fungi. In addition, we will discuss the role of sterol‐rich membrane domains in combination with cell‐end marker proteins for polarity establishment.
The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages

Mycobacterium tuberculosis generally reside in phagosomes within human macrophages that resist maturation and acidification, but exhibit significant heterogeneity. In this study we have constructed an IPTG‐inducible GFP expression system in M. tuberculosis to assess the relationship between the metabolic status of M. tuberculosis and the degree of phagosomal maturation. Using these recombinant bacteria, we have found that, in human macrophages, M. tuberculosis that respond to IPTG with expression of GFP fluorescence, and hence are metabolically active, reside in non‐acidified phagosomes that have not fused with Texas red dextran pre‐labelled lysosomes. In contrast, M. tuberculosis that fail to express GFP in response to IPTG, and hence are metabolically inactive, reside within acidified phagosomes that have fused with Texas red dextran labelled lysosomes. These studies demonstrate that metabolic activity of M. tuberculosis correlates strongly with phagosomal maturation and that the inducible GFP expression system is useful for assessing metabolic activity of intracellular M. tuberculosis.
The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum‐infected erythrocytes

During the intra‐erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein‐trafficking system beyond its confines. Membrane‐bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane‐associated histidine‐rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft‐resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1‐mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.
The type III secretion system tip complex and translocon

The type III secretion machinery of Gram‐negative bacteria, also known as the injectisome or needle complex, is composed of a basal body spanning both bacterial membranes and the periplasm, and an external needle protruding from the bacterial surface. A set of three proteins, two hydrophobic and one hydrophilic, are required to allow translocation of proteins from the bacterium to the host cell cytoplasm. These proteins are involved in the formation of a translocation pore, the translocon, in the host cell membrane. Exciting progress has recently been made on the interaction between the translocators and the injectisome needle and the assembly of the translocon in the host cell membrane. As expected, the two hydrophobic translocators insert into the target cell membrane. Unexpectedly, the third, hydrophilic translocator, forms a complex on the distal end of the injectisome needle, the tip complex, and serves as an assembly platform for the two hydrophobic translocators.
Optimizing culture conditions for high frequency somatic embryogenesis and plantlet conversion in Daucus carota L

The present investigation was carried out to establish a simple and efficient in vitro method for rapid propagation of Daucus carota L for different applications. Root, stem, leaf and seed explants were tested to induce callus on Gamborg medium supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) for cell suspension culture establishment. The leaf, stem, seed explants induced calluses were more embryogenic and viable when compared to callus derived from tap root explants. Plant regeneration potential of embryogenic callus from tap root showed very slow response producing only fewer shoots, whereas the response of other explants derived calluses like stem, leaves and seeds exhibited rapid multiple shoot formation within three weeks’ time. Tap root derived suspension culture showed more clumps with less viable embryogenic cells, in contrast to the stem and seed derived suspension cultures, where cells were more embryogenic and proliferative. Explant, concentration of carbohydrates, 2,4-D and amino acids were tested for embryo induction response and it was found that 3% sucrose, 1.0 mg/L 2,4-D and amino acid alanine had great influence on embryo induction. The somatic embryos were subjected to desiccation prior to plating on the regeneration medium. It was found that 1 h desiccation increased the regeneration percentage of the embryos and the conversion frequency of embryos to plants decreased on increased desiccation time. These optimized conditions will be very useful for studies involving genetic transformation, cell cycle, cell proliferation, synthetic seed production and other physiological studies of D. carota species.
Looking inside the box: bacterial transistor arrays

One often compares cells to computers, and signalling proteins to transistors. Location and wiring of those molecular transistors is paramount in defining the function of the subcellular chips. The bacterial chemotactic sensing apparatus is a large, stable assembly consisting of thousands of receptors, signal transducing kinases and linking proteins, and is responsible for the motile response of the bacterium to environmental signals, whether chemical, mechanical, or thermal. Because of its rich functional repertoire despite its relative simplicity, this chemosome has attracted much attention from both experimentalists and theoreticians, and the bacterial chemotaxis response becoming a benchmark in Systems Biology. Structural and functional models of the chemotactic device have been developed, often based on particular assumptions regarding the topology of the receptor lattice. In this issue of Molecular Microbiology, Briegel et al. provide a detailed view of the receptor arrangement, unravelling the wiring of the molecular signal processors.
Metabolomic profiling can predict which humans will develop liver dysfunction when deprived of dietary choline

Choline is an essential nutrient, and deficiency causes liver and muscle dysfunction. Common genetic variations alter the risk of developing organ dysfunction when choline deficient, probably by causing metabolic inefficiencies that should be detectable even while ingesting a normal choline‐adequate diet. We determined whether metabolomic profiling of plasma at baseline could predict whether humans will develop liver dysfunction when deprived of dietary choline. Fifty‐three participants were fed a diet containing 550 mg choline/70 kg/d for 10 d and then fed <50 mg choline/70 kg/d for up to 42 d. Participants who developed organ dysfunction on this diet were repleted with a choline‐adequate diet for ≥3 d. Plasma samples, obtained at baseline, end of depletion, and end of repletion, were used for targeted and nontargeted metabolomic profiling. Liver fat was assessed using magnetic resonance spectroscopy. Metabolomic profiling and targeted biochemical analyses were highly correlated for the analytes assessed by both procedures. In addition, we report relative concentration changes of other small molecules detected by the nontargeted metabolomic analysis after choline depletion. Finally, we show that metabolomic profiles of participants when they were consuming a control baseline diet could predict whether they would develop liver dysfunction when deprived of dietary choline. —Sha, W. , da Costa, K. , Fischer, L. M. , Milburn, M. V. , Lawton, K. A. , Berger, A. , Jia, W. , Zeisel, S. H. Metabolomic profiling can predict which humans will develop liver dysfunction when deprived of dietary choline. FASEB J. 24, 2962–2975 (2010). www.fasebj. org
The cia operon of Streptococcus mutans encodes a unique component required for calcium‐mediated autoregulation

Streptococcus mutans is a primary pathogen for dental caries in humans. CiaR and CiaH of S. mutans comprise a two‐component signal transduction system (TCS) involved in regulating various virulent factors. However, the signal that triggers the CiaRH response remains unknown. In this study, we show that calcium is a signal for regulation of the ciaRH operon, and that a double‐glycine‐containing small peptide encoded within the ciaRH operon (renamed ciaX) mediates this regulation. CiaX contains a serine + aspartate (SD) domain that is shared by calcium‐binding proteins. A markerless in‐frame deletion of ciaX reduced ciaRH operon expression and diminished the calcium repression of operon transcription. Point mutations of the SD domain resulted in the same phenotype as the in‐frame deletion, indicating that the SD domain is required for CiaX function. Further characterization of ciaX demonstrated that it is involved in calcium‐mediated biofilm formation. Furthermore, inactivation of ciaR or ciaH led to the same phenotype as the in‐frame deletion of ciaX, suggesting that all three genes are involved in the same regulatory pathway. Sequence analysis and real‐time RT‐PCR identified a putative CiaR binding site upstream of ciaX. We conclude that the ciaXRH operon is a three‐component, self‐regulatory system modulating cellular functions in response to calcium.
Melatonin signaling and cell protection function

Besides its well‐known regulatory role on circadian rhythm, the pineal gland hormone melatonin has other biological functions and a distinct metabolism in various cell types and peripheral tissues. In different tissues and organs, melatonin has been described to act as a paracrine and also as an intracrine and autocrine agent with overall homeostatic functions and pleiotropic effects that include cell protection and prosurvival factor. These latter effects, documented in a number of in vitro and in vivo studies, are sustained through both receptor‐dependent and ‐independent mechanisms that control detoxification and stress response genes, thus conferring protection against a number of xenobiotics and endobiotics produced by acute and chronic noxious stimuli. Redox‐sensitive components are included in the cell protection signaling of melatonin and in the resulting transcriptional response that involves the control of NF‐κB, AP‐1, and Nrf2. By these pathways, melatonin stimulates the expression of antioxidant and detoxification genes, acting in turn as a glutathione system enhancer. A further and converging mechanism of cell protection by this indoleamine described in different models seems to lie in the control of damage and signaling function of mitochondria that involves decreased production of reactive oxygen species and activation of the antiapoptotic and redox‐sensitive element Bcl2. Recent evidence suggests that upstream components in this mitochondrial route include the calmodulin pathway with its central role in melatonin signaling and the survival‐promoting component of MAPKs, ERK1/2. In this review article, we will discuss these and other molecular aspects of melatonin signaling relevant to cell protection and survival mechanisms. —Luchetti, F. , Canonico, B. , Betti, M. , Arcangeletti, M. , Pilolli, F. , Piroddi, M. , Canesi, L. , Papa, S. , Galli, F. Melatonin signaling and cell protection function. FASEB J. 24, 3603–3624 (2010). www.fasebj. org
Yeast Yak1 kinase, a bridge between PKA and stress‐responsive transcription factors, Hsf1 and Msn2/Msn4

Hsf1 and Msn2/Msn4 transcription factors in Saccharomyces cerevisiae play important roles in cellular homeostasis by activating gene expression in response to multiple stresses including heat shock, oxidative stress and nutrient starvation. Although it has been known that nuclear import of Msn2 is inhibited by PKA‐dependent phosphorylation, the mechanism for PKA‐dependent regulation of Hsf1 is not well understood. Here we demonstrate that Yak1 kinase, which is under the negative control of PKA, activates both Hsf1 and Msn2 by phosphorylation when PKA activity is lowered by glucose depletion or by overexpressing Pde2 that hydrolyses cAMP. We show that Yak1 directly phosphorylates Hsf1 in vitro, leading to the increase in DNA binding activity of Hsf1. We also demonstrate that Yak1 phosphorylates Msn2 in vitro, but does not affect DNA binding activity of Msn2 or nuclear localization of Msn2 upon glucose depletion. These results suggest a central role for Yak1 in mediating PKA‐dependent inhibition of Hsf1 and Msn2/Msn4.