The manifold roles of microbial ribosomal peptide-based natural products in physiology and ecology [Metabolism]

November 29th, 2019 by Yanyan Li, Sylvie REBUFFAT

The ribosomally synthesized and posttranslationally modified peptides (RiPPs), also called ribosomal peptide natural products (RPNPs), form a growing superfamily of natural products that are produced by many different organisms and particularly by bacteria. They are derived from precursor polypeptides whose modification by various dedicated enzymes helps establish a vast array of chemical motifs. RiPPs have attracted much interest as a source of potential therapeutic agents, and in particular as alternatives to conventional antibiotics in order to address the bacterial resistance crisis. However, their ecological roles in Nature are poorly understood and explored. The present review describes major RiPP actors in competition within microbial communities, the main ecological and physiological functions currently evidenced for RiPPs, and the microbial ecosystems that are the sites for these functions. We envision that the study of RiPPs may lead to discoveries of new biological functions and highlight that a better knowledge of how bacterial RiPPs mediate inter-/intra-species and inter-kingdom interactions will hold promise for devising alternative strategies in antibiotic development.

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules [Protein Synthesis and Degradation]

November 28th, 2019 by Friederike Hans, Hanna Glasebach, Philipp J. Kahle

Insoluble, hyperubiquitylated TAR DNA binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG forming conditions induced very strong TDP-43 ubiquitylation, insolubility and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal-regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.
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Influenza A virus-induced host caspase and viral PA-X antagonise the antiviral host factor, histone deacetylase 4 [Microbiology]

November 22nd, 2019 by Henry D Galvin, Matloob Husain

Influenza A virus (IAV) effectively manipulates host machinery to replicate. There is a growing evidence that an optimal acetylation environment in the host cell is favourable to IAV proliferation and vice versa. The histone deacetylases (HDACs) – a family of 18 host enzymes classified into four classes, are central to negatively regulating the acetylation level, hence the HDACs would not be favourable to IAV. Indeed, by using the RNA interference and overexpression strategies, we found that human HDAC4 – a class II member, possesses anti-IAV properties and is a component of host innate antiviral response. We discovered that IAV multiplication was augmented in HDAC4-depleted cells and abated in HDAC4-supplemented cells. Likewise, the expression of IFITM3, ISG15, and viperin – some of the critical markers of host anti-IAV response was abated in HDAC4-depleted cells and augmented in HDAC4-supplemented cells. In turn, IAV strongly antagonises the HDAC4, by downregulating its expression both at mRNA level via viral RNA endonuclease PA-X and at polypeptide level by inducing its cleavage via host caspase 3 in infected cells. Such HDAC4 polypeptide cleavage resulted in a ~30 kDa fragment which is also observed in some heterologous systems and may have a significant role in IAV replication.

A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration [Cell Biology]

November 20th, 2019 by Yuichi Sekine, Jane A. Lindborg, Stephen M. Strittmatter

Glial signals are known to inhibit axonal regeneration and functional recovery after mammalian central nervous system (CNS) trauma, including spinal cord injury. Such signals include membrane-associated proteins of the oligodendrocyte plasma membrane and astrocyte-derived matrix-associated proteins. Here, using cell lines and primary cortical neuron cultures, recombinant protein expression, immunoprecipitation and immunoblotting assays, transmission EM of exosomes, and axon-regeneration assays, we explored the secretion and activity of the myelin-associated, neurite outgrowth inhibitor Nogo-A and observed exosomal release of a 24-kDa, C-terminal Nogo-A fragment from cultured cells. We found that the cleavage site in this 1192-amino-acid-long fragment is located between amino acids 961–971. We also detected a Nogo-66 receptor (NgR1)-interacting Nogo-66 domain on the exosome surface. Enzyme inhibitor treatment and siRNA knockdown revealed that β-secretase 1 (BACE1) is the protease responsible for the Nogo-A cleavage. Functionally, exosomes with the Nogo-66 domain on their surface potently inhibited axonal regeneration of mechanically injured cerebral cortex neurons from mice. The production of this fragment was observed in the exosomal fraction from neuronal tissue lysates after spinal cord crush injury of mice. We also noted that relative to the exosomal marker Alix, a Nogo-immunoreactive 24-kDa protein is enriched in exosomes two-fold after injury. We conclude that membrane-associated Nogo-A produced in oligodendrocytes is processed proteolytically by BACE1, is released via exosomes, and represents a potent diffusible inhibitor of regenerative growth in NgR1-expressing axons.

The MOV10 helicase restricts hepatitis B virus replication by inhibiting viral reverse transcription [Microbiology]

November 13th, 2019 by Tingting Liu, Qingsong Sun, Yong Liu, Shan Cen, Quan Zhang

Interferons inhibit viruses by inducing antiviral protein expression. One of the interferon-induced antiviral proteins, human Moloney leukemia virus 10 (MOV10), a superfamily 1 RNA helicase, has been shown to inhibit retroviruses and several RNA viruses. However, it remains undetermined whether MOV10 also inhibits DNA viruses, including hepatitis B virus (HBV). Here, we report that MOV10 dramatically reduces the levels of intracellular HBV DNA, resulting in significant inhibition of both the HBV experimental strain and the clinical isolates. Mechanistic experiments revealed that MOV10 interacts with HBV RNA and blocks the early step of viral reverse transcription, thereby impairing viral DNA synthesis, without affecting viral gene expression and pregenomic RNA encapsidation. Moreover, mutation of the helicase domain of MOV10 caused loss of binding to HBV RNA and of the anti-HBV activity. Together, our results indicate that MOV10 restricts HBV replication, insights that may open new avenues to the development of anti-HBV therapeutics.

Biological, chemical, and biochemical strategies for modifying glycopeptide antibiotics [Enzymology]

October 31st, 2019 by Edward Marschall, Max J. Cryle, Julien Tailhades

Since the discovery of vancomycin in the 1950s, the glycopeptide antibiotics (GPAs) have been of great interest to the scientific community. These non-ribosomally biosynthesized peptides are highly crosslinked, often glycosylated, and inhibit bacterial cell wall assembly by interfering with peptidoglycan synthesis. Interest in glycopeptide antibiotics covers many scientific disciplines, due to their challenging total syntheses, complex biosynthesis pathways, mechanism of action, and high potency. After intense efforts, early enthusiasm has given way to a recognition of the challenges in chemically synthesizing GPAs and of the effort needed to study and modify GPA-producing strains to prepare new GPAs in order to address the increasing threat of microbial antibiotic resistance. Although preparation of GPAs, either by modifying the pendant groups such as saccharides or by functionalizing the N- or C-terminal moieties are readily achievable, the peptide core of these molecules – the GPA aglycone – remains highly challenging to modify. This review aims to present a comprehensive analysis of the results of GPA modification obtained with the three major approaches developed to date: in vivo strain manipulation, total chemical synthesis, and chemoenzymatic synthesis methods.

Resolving the topological enigma in Ca2+-signaling by cyclic ADP-ribose and NAADP [Signal Transduction]

October 31st, 2019 by Hon Cheung Lee, Yong Juan Zhao

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structurally distinct messengers that mobilize the endoplasmic and endo-lysosomal Ca2+-stores, respectively. Both are synthesized by CD38 molecule (CD38), which has long been thought to be a type II membrane protein whose catalytic domain, intriguingly, faces to the outside of the cell. Accordingly, for more than 20 years it has remained unresolved how CD38 can use cytosolic substrates such as NAD and NADP to produce messengers that target intracellular Ca2+-stores. The discovery of type III CD38 whose catalytic domain faces the cytosol has now begun to clarify this topological conundrum. This article reviews the ideas and clues leading to the discovery of the type III CD38; highlights an innovative approach for uncovering its natural existence, and discusses the regulators of its activity, folding and degradation. We also review the compartmentalization of cADPR and NAADP biogenesis. We further discuss the possible mechanisms that promote type III CD38 expression and appraise a proposal of a Ca2+-signaling mechanism based on substrate limitation and product translocation. The surprising finding of another enzyme that produces cADPR and NAADP, sterile alpha and TIR motif containing1 (SARM1), is described. SARM1 regulates axonal degeneration and has no sequence similarity with CD38, but can catalyze the same set of multi-reactions and has the same cytosolic orientation as the type III CD38. The intriguing finding that SARM1 is activated by nicotinamide mononucleotide to produce cADPR and NAADP suggests that it may function as a regulated Ca2+-signaling enzyme like CD38.

Detection, identification and quantification of oxidative protein modifications [Protein Synthesis and Degradation]

October 31st, 2019 by Clare L Hawkins, Michael J Davies

Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidently (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications—at the amino acid, peptide, or protein level—and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.

The scavenger receptor SCARA1(CD204) recognizes dead cells through spectrin [Immunology]

October 25th, 2019 by Chen Cheng, Zhenzheng Hu, Longxing Cao, Chao Peng, Yongning He

Scavenger receptor class A member 1 (SCARA1 or CD204) is an immune receptor highly expressed on macrophages. It forms homotrimers on cell surface and plays important roles in regulating immune responses via its involvement in multiple pathways. However, both the structure and the functional roles of SCARA1 are not fully understood. Here, we determined the crystal structure of the C-terminal SRCR domain of SCARA1 at 1.8 Å resolution, revealing its Ca2+-binding site. Results from cell-based assays revealed that SCARA1 can recognize dead cells, rather than live cells, specifically through its SRCR domain and in a Ca2+-dependent manner. Furthermore, by combining MS and biochemical assays, we found that cellular spectrin is the binding target of SCARA1 on dead cells and that the SRCR domain of SCARA1 recognizes the SPEC repeats of spectrin in the presence of Ca2+. We also found that macrophages can internalize dead cells or debris from both erythrocytes and other cells through the interaction between SCARA1 and spectrin, suggesting that SCARA1 could function as a scavenging receptor that recognizes dead cells. These results suggest that spectrin, which is one of the major components of the cytoskeleton, acts as a cellular marker that enables the recognition of dead cells by the immune system.

Intestinal breast cancer resistance protein (BCRP) requires Janus kinase 3 activity for drug efflux and barrier functions in obesity [Signal Transduction]

October 25th, 2019 by Jayshree Mishra, Randall Simonsen, Narendra Kumar

Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette (ABC) transporter proteins whose primary function is to efflux substrates bound to the plasma membrane. Impaired intestinal barrier functions play a major role in chronic low-grade inflammation (CLGI)-associated obesity, but the regulation of BCRP during obesity and its role in maintaining the intestinal barrier function during CLGI-associated obesity are unknown. In the present study, using several approaches, including efflux assays, immunoprecipitation/-blotting/-histochemistry, paracellular permeability assay, fluorescence activated cell sorting, cytokine assay, and immunofluorescence microscopy, we report that obese individuals have compromised intestinal BCRP functions and that diet-induced obese mice recapitulate these outcomes. We demonstrate that the compromised BCRP functions during obesity are due to loss of Janus kinase 3 (JAK3)-mediated tyrosine phosphorylation of BCRP. Our results indicate that JAK3-mediated phosphorylation of BCRP promotes its interactions with membrane-localized β-catenin essential not only for BCRP expression and surface localization, but also for the maintenance of BCRP-mediated intestinal drug efflux and barrier functions. We observed that reduced intestinal JAK3 expression during human obesity or JAK3 knockout in mouse or siRNA-mediated β-catenin knockdown in human intestinal epithelial cells all result in significant loss of intestinal BCRP expression and compromised colonic drug efflux and barrier functions. Our results uncover a mechanism of BCRP-mediated intestinal drug efflux and barrier functions and establish a role for BCRP in preventing CLGI-associated obesity both in humans and in mice
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