Conformational change of <Emphasis Type="Italic">Escherichia coli</Emphasis> signal recognition particle Ffh is affected by the functionality of signal peptides of ribose-binding protein

We examined the effects of synthetic signal peptides, wild-type (WT) and export-defective mutant (MT) of ribose-binding protein, on the conformational changes of signal recognition particle 54 homologue (Ffh) in Escherichia coli. Upon interaction of Ffh with WT peptide, the intrinsic Tyr fluorescence, the transition temperature of thermal unfolding, and the GTPase activity of Ffh decreased in a peptide concentration-dependent manner, while the emission intensity of 8-anilinonaphthalene-1-sulfonic acid increased. In contrast, the secondary structure of the protein was not affected. Additionally, polarization of fluorescein-labeled WT increased upon association with Ffh. These results suggest that WT peptide induces the unfolded states of Ffh. The WT-mediated conformational change of Ffh was also revealed to be important in the interaction between SecA and Ffh. However, MT had marginal effect on these conformational changes suggesting that the in vivo functionality of signal peptide is important in the interaction with Ffh and concomitant structural change of the protein.     

"Frozen" dynamic dimer model for transmembrane signaling in bacterial chemotaxis receptors.

The crystal structures of the ligand binding domain of a bacterial aspartate receptor suggest a simple mechanism for transmembrane signaling by the dimer of the receptor. On ligand binding, one domain rotates with respect to the other, and this rotational motion is proposed to be transmitted through the membrane to the cytoplasmic domains of the receptor.     

Two-component signal transduction systems and regulation of virulence factors in <Emphasis Type="Italic">Xanthomonas</Emphasis>: a perspective

Two-component signal transduction systems (TCSTSs), consisting of a histidine kinase and a response regulator, play a critical role in regulating virulence gene expression in Gram-negative phytopathogenic bacteria Xanthomonas spp.. To date, 12 TCSTS genes have been identified, accounting for approximately 10% of the TCSTS genes in each genome that have been experimentally identified to be related to pathogenesis. These TCSTSs modulate the expression of a number of virulence factors through diverse molecular mechanisms such as interacting with DNA, protein-binding and involvement in second messenger metabolism, which generates a high level of regulatory versatility. Here we summarize the current knowledge in this field and discuss the emerging themes and remaining questions that are important in deciphering the signaling network of TCSTSs in Xanthomonas.     

Fluorescence spectroscopy of soluble E. coli SPase I Δ2‐75 reveals conformational changes in response to ligand binding

The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane‐embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2‐75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2‐75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane‐interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the K_d determined to be 4.4 μM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids. Proteins 2014; 82:596–606. © 2013 Wiley Periodicals, Inc.     

Let's Call A Truce…For Now: The Silent Bared‐Teeth Face Expression in Mandrills (Mandrillus sphinx) During Baseline and Post‐Conflict Conditions

The distinction between signals that are friendly and those that are non‐aggressive but motivationally neutral (signals of benign intent, SBIs) has not often been well elucidated in the literature. Although both signals occur in similar contexts, friendly signals should be exchanged more often between animals with good relationships whereas SBIs should be more commonly exchanged between animals with poor or unpredictable relationships. The importance of this distinction is particularly salient in the post‐conflict context, because the two different types of signals may have disparate distributions and functions. This study examines the nature of the silent bared‐teeth face (SBTF) in captive mandrills (Mandrillus sphinx) both during baseline interactions and following aggressive conflicts. We report that the SBTF is most commonly exchanged between mandrills with high rates of agonism. In addition, the SBTF is the most common post‐conflict signal exchanged, and the mandrills exchanging this signal after fighting also have poor relationships. We conclude that, although one must be careful in generalizing from studies of captive populations, the mandrill SBTF observed in this study is most accurately classified as a signal of benign intent, but not a truly friendly signal and discuss problems with interpreting post‐conflict data without distinguishing between these types of signals.     

Interferon‐γ induces leucine‐rich repeat kinase LRRK2 via extracellular signal‐regulated kinase ERK5 in macrophages

The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.

     

Alteration of biomass and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots by media sterilization method and sugars

Transformed root cultures of Artemisia annua grown in autoclaved medium show large variations in biomass and artemisinin production regardless of the culture conditions or clonal type. However, using filter-sterilized sugars singly or in combination while holding the carbon level in the medium constant resulted in an unexpected variability in biomass production and artemisinin yield. Autoclaving results in variable hydrolysis of sucrose in the culture medium. Subsequent experiments using combinations of filter-sterilized sugars at a constant total carbon level in the medium showed a stimulation of artemisinin production by glucose. Growth in sucrose was equivalent to growth in fructose and significantly better than in glucose. These results suggest that sugars may be affecting terpenoid metabolism not only as carbon sources, but also as signal molecules.     

A candidate packaging signal of human rotavirus differentiating Wa‐like and DS‐1‐like genomic constellations

Rotavirus A (RVA) possesses a genome of 11 segmented RNAs. In human RVA, two major genomic constellations are represented by prototype strains Wa and DS‐1. Here packaging signals differentiating Wa‐like and DS‐1‐like genomic constellations were searched for by analyzing genomic sequences of Wa‐like and DS‐1‐like strains. One pair of 11 nucleotide sites in the coding regions of viral structural protein (VP) 2 and VP6 was found to be complementary specifically among Wa‐like strains. These sites tended to be free from base‐pairing in secondary structures of genomic segments, suggesting that they may serve as a packaging signal in Wa‐like strains.     

Phosphorylation‐dependent Akt–Inversin interaction at the basal body of primary cilia

A primary cilium is a microtubule‐based sensory organelle that plays an important role in human development and disease. However, regulation of Akt in cilia and its role in ciliary development has not been demonstrated. Using yeast two‐hybrid screening, we demonstrate that Inversin (INVS) interacts with Akt. Mutation in the INVS gene causes nephronophthisis type II (NPHP2), an autosomal recessive chronic tubulointerstitial nephropathy. Co‐immunoprecipitation assays show that Akt interacts with INVS via the C‐terminus. In vitro kinase assays demonstrate that Akt phosphorylates INVS at amino acids 864–866 that are required not only for Akt interaction, but also for INVS dimerization. Co‐localization of INVS and phosphorylated form of Akt at the basal body is augmented by PDGF‐AA. Akt‐null MEF cells as well as siRNA‐mediated inhibition of Akt attenuated ciliary growth, which was reversed by Akt reintroduction. Mutant phosphodead‐ or NPHP2‐related truncated INVS, which lack Akt phosphorylation sites, suppress cell growth and exhibit distorted lumen formation and misalignment of spindle axis during cell division. Further studies will be required for elucidating functional interactions of Akt–INVS at the primary cilia for identifying the molecular mechanisms underlying NPHP2.     

Antigen‐responsive regulation of Cell motility and migration via the signalobodies based on c‐Fms and c‐Mpl

Since cell migration plays critical roles in development and homeostasis of the body, artificial control of cell migration would be promising for the treatment of various diseases related to migration. To this end, we previously developed single‐chain Fv (scFv)/receptor chimeras, named signalobodies, which can control cell fates via a specific antigen that is different from natural cytokines. Although a conventional chemotaxis chamber assay revealed that several signalobodies based on receptor tyrosine kinases transduced antigen‐dependent migration signals, we have never performed direct observation of the cells to obtain more information on overall properties of cell motility and migration. In this study, we utilized murine pro‐B Ba/F3 cells expressing either a scFv‐Fms or scFv‐Mpl signalobody, and compared their migratory characteristics. We employed a lipid–polyethylene glycol conjugate to softly immobilize the suspension cells on a slide, which facilitated direct observation of chemokinetic activity of the cells. Consequently, both cells markedly exhibited chemokinesis in response to a specific antigen. In addition, the cells were subjected to a stable antigen‐concentration gradient to observe horizontal directional cell migration in real time. The results showed that the cells expressing scFv‐Fms underwent directional migration toward a positive antigen‐concentration gradient. Taken together, we successfully demonstrated antigen‐responsive regulation of cell motility and migration via the signalobodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:411–417, 2014     

Intracellular signalling proteins as ‘smart’ agents in parallel distributed processes

In eucaryotic organisms, responses to external signals are mediated by a repertoire of intracellular signalling pathways that ultimately bring about the activation/inactivation of protein kinases and/or protein phosphatases. Until relatively recently, little thought had been given to the intracellular distribution of the components of these signalling pathways. However, experimental evidence from a diverse range of organisms indicates that rather than being freely distributed, many of the protein components of signalling cascades show a significant degree of spatial organisation. Here, we briefly review the roles of ‘anchor’, ‘scaffold’ and ‘adaptor’ proteins in the organisation and functioning of intracellular signalling pathways. We then consider some of the parallel distributed processing capacities of these adaptive systems. We focus on signalling proteins-both as individual ‘devices’ (agents) and as ‘networks’ (ecologies) of parallel processes. Signalling proteins are described as ‘smart thermodynamic machines’ which satisfy ‘gluing’ (functorial) roles in the information economy of the cell. This combines two information-processing views of signalling proteins. Individually, they show ‘cognitive’ capacities and collectively they integrate (cohere) cellular processes. We exploit these views by drawing comparisons between signalling proteins and verbs. This text/dialogical metaphor also helps refine our view of signalling proteins as context-sensitive information processing agents.     

Engineered single‐ and multi‐cell chemotaxis pathways in E. coli

We have engineered the chemotaxis system of Escherichia coli to respond to molecules that are not attractants for wild‐type cells. The system depends on an artificially introduced enzymatic activity that converts the target molecule into a ligand for an E. coli chemoreceptor, thereby enabling the cells to respond to the new attractant. Two systems were designed, and both showed robust chemotactic responses in semisolid and liquid media. The first incorporates an asparaginase enzyme and the native E. coli aspartate receptor to produce a response to asparagine; the second uses penicillin acylase and an engineered chemoreceptor for phenylacetic acid to produce a response to phenylacetyl glycine. In addition, by taking advantage of a ‘hitchhiker’ effect in which cells producing the ligand can induce chemotaxis of neighboring cells lacking enzymatic activity, we were able to design a more complex system that functions as a simple microbial consortium. The result effectively introduces a logical ‘AND’ into the system so that the population only swims towards the combined gradients of two attractants.     

Hydrogen sulphide decreases IL‐1β‐induced activation of fibroblast‐like synoviocytes from patients with osteoarthritis

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H₂S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H₂S on fibroblast‐like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro‐inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL‐1β and treated with the H₂S donor NaHS. Cellular responses were analysed by ELISA, quantitative real‐time PCR, phospho‐MAPkinase array and Western blotting. Treatment‐induced effects on cellular structure and synovial architecture were investigated in three‐dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL‐1β‐induced secretion of IL‐6, IL‐8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo‐proteinases MMP‐2 and MMP‐14. IL‐1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL‐1β‐induced activation of several MAPK whereas it increased phosphorylation of pro‐survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer‐like structure; stimulation with IL‐1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H₂S partially antagonizes IL‐1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.     

Lipoprotein trafficking in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane. In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken. Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE). The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined. On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins. We also discuss why inner membrane-specific lipoproteins remain on the inner membrane.     

Male adaptations to minimize sexual cannibalism during reproduction in the funnel‐web spider Hololena curta

Males of many spider species risk being attacked and cannibalized while searching for, courting, and mating with conspecific females. However, there are exceptions. We show that the funnel‐web spider, Hololena curta, has 3 adaptations that minimize risk to males during courtship and mating, and enhance reproductive success. First, males detected chemical or tactile signals associated with webs of virgin females, and differentiated them from webs of mated females, enabling males to increase encounter rates with virgin females and avoid aggressive mated females. Second, males produced stereotyped vibrational signals during courting which induced female quiescence and suppressed female aggression. Third, when touched by males, sexually receptive females entered a cataleptic state, allowing males to safely approach and copulate. Because males can mate multiple times and the sex ratio in natural populations of H. curta is female biased, overall reproductive output is likely increased by males of this species avoiding sexual cannibalism.     

β‐arrestin‐1 mediates the TCR‐triggered re‐routing of distal receptors to the immunological synapse by a PKC‐mediated mechanism

T‐cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen‐presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β‐arrestin‐1 as a ligand of non‐phosphorylated resting TCRs. Using dominant‐negative and knockdown approaches we demonstrate that β‐arrestin‐1 is required for the internalization and downregulation of non‐engaged bystander TCRs. Furthermore, TCR triggering provokes the β‐arrestin‐1‐mediated downregulation of the G‐protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β‐arrestin‐1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR‐triggered PKC‐mediated phosphorylation of β‐arrestin‐1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.     

Circadian rhythms—mammalian aspects

Across a large phylogenetic distance, circadian systems that regulate daily changes in physiology and behavior exhibit fundamental properties that are remarkably similar, suggesting either a conservation or constraint on the optimal organization of the biological clock. Whether or not this is a reflection of homologies in the genetic underpinnings of these clocks is unknown. Nonetheless, evidence for comparable organization from genetic, pharmacological and behavioral studies exists for diverse species. This allows insights into circadian organization to be drawn from a variety of models that provide access at different levels. Historically, mammalian models have been particularly useful for describing fundamental properties of clocks and their regulatory mechanisms. Recent discoveries of clock mutations in hamsters and mice will provide new opportunities for comprehensive studies involving multiple levels of organization