ResearchPad - 34 https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Membrane association but not identity is required for LRRK2 activation and phosphorylation of Rab GTPases]]> https://www.researchpad.co/article/elastic_article_15315 LRRK2 kinase mutations cause familial Parkinson’s disease and increased phosphorylation of a subset of Rab GTPases. Rab29 recruits LRRK2 to the trans-Golgi and activates it there, yet some of LRRK2’s major Rab substrates are not on the Golgi. We sought to characterize the cell biology of LRRK2 activation. Unlike other Rab family members, we show that Rab29 binds nucleotide weakly, is poorly prenylated, and is not bound to GDI in the cytosol; nevertheless, Rab29 only activates LRRK2 when it is membrane bound and GTP bound. Mitochondrially anchored, GTP-bound Rab29 is both a LRRK2 substrate and activator, and it drives accumulation of active LRRK2 and phosphorylated Rab10 on mitochondria. Importantly, mitochondrially anchored LRRK2 is much less capable of phosphorylating plasma membrane–anchored Rab10 than soluble LRRK2. These data support a model in which LRRK2 associates with and dissociates from distinct membrane compartments to phosphorylate Rab substrates; if anchored, LRRK2 can modify misdelivered Rab substrates that then become trapped there because GDI cannot retrieve them.

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<![CDATA[Higher-order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes]]> https://www.researchpad.co/article/N8aa28941-0406-4cfc-ada2-7f301bbf740f

Endosomal maturation and distribution, driven by membrane remodeling, are critical for receptor traffic and signaling. Using both in vitro and in vivo approaches, Wang et al. reveal an unexpected coiled-coil–mediated membrane remodeling activity of SNX16 that controls neuronal endosomal tubulation, distribution, and receptor traffic.

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<![CDATA[LTK is an ER-resident receptor tyrosine kinase that regulates secretion]]> https://www.researchpad.co/article/Nb241a4e3-9203-4bab-87dd-85c699f4a230

The endoplasmic reticulum (ER) is a major regulator of cellular proteostasis. However, only little is known about signaling molecules resident to this organelle. Centonze et al. identify LTK as the first ER-resident receptor tyrosine kinase and show that it stimulates secretory trafficking out of the ER.

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<![CDATA[Synaptic neurexin-1 assembles into dynamically regulated active zone nanoclusters]]> https://www.researchpad.co/article/N2375b59c-7988-4c48-9a30-d168163c3153

Using super-resolution microscopy, Trotter et al. illuminate an unexpected nanoscale organization of excitatory synapses in which neurexins are assembled in the presynaptic active zone into dynamic nanoclusters that are regulated by ADAM10-mediated ectodomain cleavage.

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<![CDATA[Influenza virus N-linked glycosylation and innate immunity]]> https://www.researchpad.co/article/5c4b941ed5eed0c48487de5a

Influenza viruses cause seasonal epidemics and sporadic pandemics in humans. The virus’s ability to change its antigenic nature through mutation and recombination, and the difficulty in developing highly effective universal vaccines against it, make it a serious global public health challenge. Influenza virus’s surface glycoproteins, hemagglutinin and neuraminidase, are all modified by the host cell’s N-linked glycosylation pathways. Host innate immune responses are the first line of defense against infection, and glycosylation of these major antigens plays an important role in the generation of host innate responses toward the virus. Here, we review the principal findings in the analytical techniques used to study influenza N-linked glycosylation, the evolutionary dynamics of N-linked glycosylation in seasonal versus pandemic and zoonotic strains, its role in host innate immune responses, and the prospects for lectin-based therapies. As the efficiency of innate immune responses is a critical determinant of disease severity and adaptive immunity, the study of influenza glycobiology is of clinical as well as research interest.

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<![CDATA[Synthetic bovine lactoferrin peptide Lfampin kills Entamoeba histolytica trophozoites by necrosis and resolves amoebic intracecal infection in mice]]> https://www.researchpad.co/article/5c4b940fd5eed0c48487dbec

Amoebiasis caused by the protozoan parasite Entamoeba histolytica remains a public health problem in developing countries, making the identification of new anti-amoebic compounds a continuing priority. Previously, we have shown that lactoferrin (Lf) and several Lf-derived peptides exhibit in vitro anti-amoebic activity independently of their iron-binding activity. Here, we evaluated the amoebicidal effect of synthetic Lf-derived peptides Lfcin-B, Lfcin 17-30, and Lfampin, analyzed the mechanism of death induced by the peptides and determined their therapeutic effects on murine intestinal amoebiasis. MTT assays in trophozoite cultures of E. histolytica exposed to each peptide (1–1000 μM) showed that Lfampin is far more amoebicidal than Lfcins. Lfampin killed 80% of trophozoites at doses higher than 100 μM in 24 h, and FACs analysis using Annexin V/propidium iodide showed that death occurred mainly by necrosis. In contrast, Lfcin-B and Lfcin 17-30 appeared to have no significant effect on amoebic viability. FACs and confocal microscopy analysis using FITC-labeled peptides showed that all three peptides are internalized by the amoeba mainly using receptor (PI3K signaling) and actin-dependent pathways but independent of clathrin. Docking studies identified cholesterol in the amoeba’s plasma membrane as a possible target of Lfampin. Oral treatment of intracecally infected mice with the abovementioned peptides at 10 mg/kg for 4 days showed that Lfampin resolved 100% of the cases of intestinal amoebiasis, whereas Lfcin 17-30 and Lfcin-B were effective in resolving infection in 80 and 70% of cases, respectively. These data show that although synthetic bovine Lf-derived peptides exhibit varying amoebicidal potentials in vitro, they do resolve murine intestinal amoebiasis efficiently, suggesting that they may be useful as a therapeutic treatment.

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<![CDATA[The state of art of neutrophil extracellular traps in protozoan and helminthic infections]]> https://www.researchpad.co/article/5c4b93edd5eed0c48487d506

Neutrophil extracellular traps (NETs) are DNA fibers associated with histones, enzymes from neutrophil granules and anti-microbial peptides. NETs are released in a process denominated NETosis, which involves sequential steps that culminate with the DNA extrusion. NETosis has been described as a new mechanism of innate immunity related to defense against different pathogens. The initial studies of NETs were carried out with bacteria and fungi, but currently a large variety of microorganisms capable of inducing NETs have been described including protozoan and helminth parasites. Nevertheless, we have little knowledge about how NETosis process is carried out in response to the parasites, and about its implication in the resolution of this kind of disease. In the best case, the NETs entrap and kill parasites in vitro, but in others, immobilize the parasites without affecting their viability. Moreover, insufficient studies on the NETs in animal models of infections that would help to define their role, and the association of NETs with chronic inflammatory pathologies such as those occurring in several parasitic infections have left open the possibility of NETs contributing to pathology instead of protection. In this review, we focus on the reported mechanisms that lead to NET release by protozoan and helminth parasites and the evidence that support the role of NETosis in the resolution or pathogenesis of parasitic diseases.

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<![CDATA[Specificity and mechanism of carbohydrate demethylation by cytochrome P450 monooxygenases]]> https://www.researchpad.co/article/5c2d429ed5eed0c484e05acd

Degradation of carbohydrates by bacteria represents a key step in energy metabolism that can be inhibited by methylated sugars. Removal of methyl groups, which is critical for further processing, poses a biocatalytic challenge because enzymes need to overcome a high energy barrier. Our structural and computational analysis revealed how a member of the cytochrome P450 family evolved to oxidize a carbohydrate ligand. Using structural biology, we ascertained the molecular determinants of substrate specificity and revealed a highly specialized active site complementary to the substrate chemistry. Invariance of the residues involved in substrate recognition across the subfamily suggests that they are critical for enzyme function and when mutated, the enzyme lost substrate recognition. The structure of a carbohydrate-active P450 adds mechanistic insight into monooxygenase action on a methylated monosaccharide and reveals the broad conservation of the active site machinery across the subfamily.

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<![CDATA[Mast cell deficiency in mice results in biomass overgrowth and delayed expulsion of the rat tapeworm Hymenolepis diminuta]]> https://www.researchpad.co/article/5c1c2c55d5eed0c4844609fa

Infection with helminth parasites evokes a complex cellular response in the host, where granulocytes (i.e. eosinophils, basophils and mast cells (MCs)) feature prominently. In addition to being used as markers of helminthic infections, MCs have been implicated in worm expulsion since animals defective in c-kit signaling, which results in diminished MC numbers, can have delayed worm expulsion. The role of MCs in the rejection of the rat tapeworm, Hymenolepsis diminuta, from the non-permissive mouse host is not known. MC-deficient mice display a delay in the expulsion of H. diminuta that is accompanied by a less intense splenic Th2 response, as determined by in vitro release of interleukin (IL)-4, IL-5 and IL-13 cytokines. Moreover, worms retrieved from MC-deficient mice were larger than those from wild-type (WT) mice. Assessment of gut-derived IL-25, IL-33, thymic stromal lymphopoietin revealed lower levels in uninfected MC-deficient mice compared with WT, suggesting a role for MCs in homeostatic control of these cytokines: differences in these gut cytokines between the mouse strains were not observed after infection with H. diminuta. Finally, mice infected with H. diminuta display less severe dinitrobenzene sulphonic acid (DNBS)-induced colitis, and this beneficial effect of the worm was unaltered in MC-deficient mice challenged with DNBS, as assessed by a macroscopic disease score. Thus, while MCs are not essential for rejection of H. diminuta from mice, their absence slows the kinetics of expulsion allowing the development of greater worm biomass prior to successful rejection of the parasitic burden.

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<![CDATA[Fantastic voyage: the journey of intestinal microbiota-derived microvesicles through the body]]> https://www.researchpad.co/article/5c114487d5eed0c4845d3ede

As part of their life cycle, Gram-negative bacteria produce and release microvesicles (outer membrane vesicles, OMVs) consisting of spherical protrusions of the outer membrane that encapsulate periplasmic contents. OMVs produced by commensal bacteria in the gastrointestinal (GI) tract of animals are dispersed within the gut lumen with their cargo and enzymes being distributed across and throughout the GI tract. Their ultimate destination and fate is unclear although they can interact with and cross the intestinal epithelium using different entry pathways and access underlying immune cells in the lamina propria. OMVs have also been found in the bloodstream from which they can access various tissues and possibly the brain. The nanosize and non-replicative status of OMVs together with their resistance to enzyme degradation and low pH, alongside their ability to interact with the host, make them ideal candidates for delivering biologics to mucosal sites, such as the GI and the respiratory tract. In this mini-review, we discuss the fate of OMVs produced in the GI tract of animals with a focus on vesicles released by Bacteroides species and the use of OMVs as vaccine delivery vehicles and other potential applications.

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<![CDATA[Do nitric oxide, carbon monoxide and hydrogen sulfide really qualify as ‘gasotransmitters’ in bacteria?]]> https://www.researchpad.co/article/5c114489d5eed0c4845d3f4a

A gasotransmitter is defined as a small, generally reactive, gaseous molecule that, in solution, is generated endogenously in an organism and exerts important signalling roles. It is noteworthy that these molecules are also toxic and antimicrobial. We ask: is this definition of a gasotransmitter appropriate in the cases of nitric oxide, carbon monoxide and hydrogen sulfide (H2S) in microbes? Recent advances show that, not only do bacteria synthesise each of these gases, but the molecules also have important signalling or messenger roles in addition to their toxic effects. However, strict application of the criteria proposed for a gasotransmitter leads us to conclude that the term ‘small molecule signalling agent’, as proposed by Fukuto and others, is preferable terminology.

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<![CDATA[New tools for “hot-wiring” clathrin-mediated endocytosis with temporal and spatial precision]]> https://www.researchpad.co/article/5c01f904d5eed0c4842bf905

Wood et al. have developed inducible chemical and optogenetic systems for triggering clathrin-mediated endocytosis using minimal clathrin-binding domains. Bypassing regulatory steps in vesicle creation will enable a better understanding of the factors required for vesicle initiation and processing, which the authors use to define how clathrin functionally interacts with its adaptor AP2.

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<![CDATA[Ragulator—a multifaceted regulator of lysosomal signaling and trafficking]]> https://www.researchpad.co/article/5c01f925d5eed0c4842c03ea

Colaço and Jäättelä discuss studies from Pu et al. and Filipek et al. that identify Ragulator as a dynamic regulator of lysosomal trafficking.

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<![CDATA[Segregation in the Golgi complex precedes export of endolysosomal proteins in distinct transport carriers]]> https://www.researchpad.co/article/5c01f91fd5eed0c4842c01a1

Chen et al. present evidence that two sets of newly synthesized endolysosomal proteins segregate in the Golgi complex before their export in two distinct populations of transport carriers, by mechanisms that are respectively dependent or independent of sorting signal–adaptor interactions.

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<![CDATA[cTAGE5 deletion in pancreatic β cells impairs proinsulin trafficking and insulin biogenesis in mice]]> https://www.researchpad.co/article/5c01f91cd5eed0c4842c0087

In this study, Fan et al. show that cTAGE5 interacts with the v-SNARE Sec22b to regulate proinsulin processing and COPII-dependent trafficking from the ER to the Golgi, thereby influencing glucose tolerance.

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<![CDATA[The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network]]> https://www.researchpad.co/article/5c01f8fdd5eed0c4842bf6df

The human pathogen Toxoplasma sequesters host vesicles filled with nutrients into its parasitophorous vacuole. Romano et al. now identify parasite effectors located at an intravacuolar membrane network that control internalization and degradation of these vesicles and cargo release within the vacuole.

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<![CDATA[Feedback inhibition of actin on Rho mediates content release from large secretory vesicles]]> https://www.researchpad.co/article/5c129a84d5eed0c4848c8401

This work identified a cycle of actin assembly and disassembly in large secretory vesicles of Drosophila salivary glands. Actin disassembly is triggered by actin-dependent recruitment of a RhoGAP protein and is essential for the contractility of the vesicle, leading to content release to the lumen.

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<![CDATA[The synergistic antimicrobial effects of novel bombinin and bombinin H peptides from the skin secretion of Bombina orientalis]]> https://www.researchpad.co/article/5bf44cf9d5eed0c4846e3588

Bombinin and bombinin H are two antimicrobial peptide (AMP) families initially discovered from the skin secretion of Bombina that share the same biosynthetic precursor-encoding cDNAs, but have different structures and physicochemical properties. Insight into their possible existing relationship lead us to perform the combination investigations into their anti-infectious activities. In this work, we report the molecular cloning and functional characterization of two novel AMPs belonging to bombinin and bombinin H families from secretions of Bombina orientalis. Their mature peptides (BHL-bombinin and bombinin HL), coded by single ORF, were chemically synthesized along with an analogue peptide that replaced L-leucine with D-leucine from the second position of the N-terminus (bombinin HD). CD analysis revealed that all of them displayed well-defined α-helical structures in membrane mimicking environments. Furthermore, BHL-bombinin displayed broad-spectrum bactericidal activities on a wide range of microorganisms, while bombinin H only exhibited a mildly bacteriostatic effect on the Gram-positive bacteria Staphylococcus aureus. The combination potency of BHL-bombinin with either bombinin HL or bombinin HD showed the synergistic inhibition activities against S. aureus (fractional inhibitory concentration index (FICI): 0.375). A synergistic effect has also been observed between bombinin H and ampicillin, which was further systematically evaluated and confirmed by in vitro time-killing investigations. Haemolytic and cytotoxic examinations exhibited a highly synergistic selectivity and low cytotoxicity on mammalian cells of these three peptides. Taken together, the discovery of the potent synergistic effect of AMPs in a single biosynthetic precursor with superior functional selectivity provides a promising strategy to combat multidrug-resistant pathogens in clinical therapy.

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