Pro‐apoptotic activity of mBNIP‐21 depends on its BNIP‐2 and Cdc42GAP homology (BCH) domain and is enhanced by coxsackievirus B3 infection

Our previous study reported that mouse BNIP‐21 (mBNIP‐21) induces apoptosis through a mitochondria‐dependent pathway. To map the functional domains of mBNIP‐21, we performed mutational analyses and demonstrated that the BNIP‐2 and Cdc42GAP homology (BCH) domain is required for apoptosis induction by mBNIP‐21 targeting the mitochondria and inducing cytochrome c release. This pro‐apoptotic activity was enhanced by coxsackievirus infection. However, deletion of the Bcl‐2 homology 3 (BH3)‐like domain, a well‐known cell ‘death domain’ in proapoptotic Bcl‐2 family proteins, did not affect the activity of mBNIP‐21. These data were further supported by transfection of a mouse Bax (mBax) mutant, whose BH3 was replaced by the mBNIP‐21 BH3‐like domain. This replacement significantly reduced the pro‐apoptotic activity of mBax. We also found that the predicted calcium binding domain has no contribution to the mBNIP‐21‐induced apoptosis. Further mapping of the motifs of BCH domain demonstrated that deletion of the hydrophobic motif proximal to the C‐terminal of the BCH significantly reduced its proapoptotic activity. These findings suggest that mBNIP‐21, as a member of the BNIP subgroup of the Bcl‐2‐related proteins, functions without need of BH3 but its BCH domain is critical for its activity in inducing cell elongation, membrane protrusions and apoptotic cell death.     

BDNF‐mediated migration of cardiac microvascular endothelial cells is impaired during ageing

This study indicates that brain‐derived neurotrophic factor (BDNF) can promote young cardiac microvascular endothelial cells (CMECs) to migrate via the activation of the BDNF‐TrkB‐FL‐PI3K/Akt pathway, which may benefit angiogenesis after myocardial infarction (MI). However, the ageing of CMECs led to changes in the expression of receptor Trk isoforms in that among the three isoforms (TrkB‐FL, TrkB‐T1 and TrkB‐T2), only one of its truncated isoforms, TrkB‐T1, continued to be expressed, which leads to the dysfunction of its ligand, a decrease in the migration of CMECs and increased injury in ageing hearts. This shift in receptor isoforms in aged CMECs, together with changes in the ageing microenvironment, might predispose ageing hearts to decreased angiogenic potential and increased cardiac pathology.     

Isopropyl‐phloroglucinol‐DHA protects outer retinal cells against lethal dose of all‐trans‐retinal

All‐trans‐retinal (atRAL) is a highly reactive carbonyl specie, known for its reactivity on cellular phosphatidylethanolamine in photoreceptor. It is generated by photoisomerization of 11‐cis‐retinal chromophore linked to opsin by the Schiff's base reaction. In ABCA4‐associated autosomal recessive Stargardt macular dystrophy, atRAL results in carbonyl and oxidative stress, which leads to bisretinoid A2E, accumulation in the retinal pigment epithelium (RPE). This A2E‐accumulation presents as lipofuscin fluorescent pigment, and its photooxidation causes subsequent damage. Here we describe protection against a lethal dose of atRAL in both photoreceptors and RPE in primary cultures by a lipidic polyphenol derivative, an isopropyl‐phloroglucinol linked to DHA, referred to as IP‐DHA. Next, we addressed the cellular and molecular defence mechanisms in commonly used human ARPE‐19 cells. We determined that both polyunsaturated fatty acid and isopropyl substituents bond to phloroglucinol are essential to confer the highest protection. IP‐DHA responds rapidly against the toxicity of atRAL and its protective effect persists. This healthy effect of IP‐DHA applies to the mitochondrial respiration. IP‐DHA also rescues RPE cells subjected to the toxic effects of A2E after blue light exposure. Together, our findings suggest that the beneficial role of IP‐DHA in retinal cells involves both anti‐carbonyl and anti‐oxidative capacities.     

Engineering a G protein‐coupled receptor for structural studies: Stabilization of the BLT1 receptor ground state

Structural characterization of membrane proteins is hampered by their instability in detergent solutions. We modified here a G protein‐coupled receptor, the BLT1 receptor of leukotriene B₄, to stabilize it in vitro. For this, we introduced a metal‐binding site connecting the third and sixth transmembrane domains of the receptor. This modification was intended to restrain the activation‐associated relative movement of these helices that results in a less stable packing in the isolated receptor. The modified receptor binds its agonist with low‐affinity and can no longer trigger G protein activation, indicating that it is stabilized in its ground state conformation. Of importance, the modified BLT1 receptor displays an increased temperature‐, detergent‐, and time‐dependent stability compared with the wild‐type receptor. These data indicate that stabilizing the ground state of this GPCR by limiting the activation‐associated movements of the transmembrane helices is a way to increase its stability in detergent solutions; this could represent a forward step on the way of its crystallization.     

Life‐history traits of temperate and thermophilic barracudas (Teleostei: Sphyraenidae) in the context of sea warming in the Mediterranean Sea

This study indicated that the life‐history traits of European barracuda Sphyraena sphyraena are apparently better suited to their environmental conditions compared to the more physically restricted life‐history traits of the yellow‐mouth barracuda Sphyraena viridensis, which co‐habit the north‐western Mediterranean Sea. The latter thermophilic species has a considerably higher reproductive potential as it invests its energy reserves in larger numbers of hydrated eggs per spawning batch. This would favour its population growth rates within the study area, especially if sea warming continues, in which case it is likely that the spawning phenology of this species would give it an advantage.     

Molecularly modified VP3 (30–121) induces apoptosis in human bladder cancer (EJ) cells but not in normal (3T3) cells

The chicken anaemia virus protein 3 (VP3 or apoptin) induces apoptosis specifically in tumour and transformed cells but not in normal cells. This selective apoptosis is ideal for therapeutic purposes. However, VP3, a heterologous protein, is immunogenic in vivo. Such a drawback limits its clinical usage. To diminish its potential immunogenicity, we modified the sequence of VP3. The VP3 genes functional sequence starts at 33 AA (amino acids) from the N‐terminal side, and the first protein structural domain consists of 30 AA. We found that the first domain of VP3 contains no functional sequences. Therefore, this domain was removed to test whether its absence affects apoptosis. Transfection of EGFP (enhance green fluorescent protein)‐modified‐VP3 or HA‐modified‐VP3 in bladder cancer cell lines (EJ) resulted in its expression, successful localization to the nucleus and efficient induction of apoptosis. Expression of EGFP‐modified‐VP3 or HA‐modified‐VP3 had no influence on mouse fibroblast cells (3T3). The modified VP3 (30–121), like the wild‐type VP3, induced EJ cell apoptosis without affecting 3T3 cells. This study increases our understanding of modified VP3 (30–121) as a possible substitute for the wild‐type VP3, which makes VP3 (30–121) an interesting candidate for the development of novel therapeutic strategies.     

miR‐18a promotes Mycobacterial survival in macrophages via inhibiting autophagy by down‐regulation of ATM

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of leading causes of global deaths. This study aimed to explore the role of miR‐18a in RAW264.7 cells response to Mtb infection. Exosomes derived from Mtb‐infected cells were isolated and further validated by size, transmission electron microscopy and Western blot. RT‐PCR was utilized to measure miR‐18a expression. Cell viability and ultrastructure were examined by CFU counting, CCK‐8 and electron microscope, respectively. Potential target genes of miR‐18a were predicted with bioinformatics and further confirmed using RT‐PCR, Western blot and laser confocal microscope analysis, respectively. LC3, AMPK and mTOR were measured using Western blot. We found that miR‐18a was induced both in Mtb‐infected RAW264.7 cells and its derived exosomes compared with the controls. In addition, up‐regulation of miR‐18a promoted intracellular Mtb survival, attenuated cell viability and reduced LC3‐II level, while its down‐regulation had the opposite effect. miR‐18a overexpression suppressed level of ATM, one possible target of miR‐18a, while its underexpression enhanced ATM. We also found that inhibition of ATM induced LC3‐II decrease in Mtb‐infected cells and could reverse the increase of LC3‐II caused by inhibition of miR‐18a. Moreover, down‐regulation of miR‐18a increased p‐AMPK level while reduction of ATM could reverse the change. Taken together, our results suggest that miR‐18a is up‐regulated in macrophages response to Mtb infection, and it promotes intracellular Mtb survival through repressing autophagic process by down‐regulation of ATM pathway. This provides new thought for TB pathogenesis, diagnosis and treatment.     

Camptotheca acuminata 10‐hydroxycamptothecin O‐methyltransferase: an alkaloid biosynthetic enzyme co‐opted from flavonoid metabolism

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10‐hydroxycamptothecin, and 10‐methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10‐hydroxycamptothecin O‐methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A‐ring 7‐OH of flavonoids, which is structurally equivalent to the 10‐OH of 10‐hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3‐D positioning of the 7‐OH, A‐ and C‐rings of flavonoids is nearly identical to the 10‐OH, A‐ and B‐rings of 10‐hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10‐hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7‐OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10‐hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non‐inhibitory flavonoid glycosides.     

Srs2 mediates PCNA‐SUMO‐dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S‐PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S‐PCNA and Srs2 block the synthesis‐dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross‐over. This new Srs2 activity requires the SUMO interaction motif at its C‐terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S‐PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1‐dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.     

Passive Membrane Penetration of the Serotonin Precursor 5‐Hydroxytryptophan is Controlled by Its Zwitterion

Species‐specific partition coefficients in the octanol/water system were determined for the neurotransmitter serotonin (5‐HT) and its precursor 5‐hydroxytryptophan (5‐HTP). The pH‐independent partition coefficients (p) of the individual microspecies were determined by combination of experimentally measured distribution constants and a custom‐tailored evaluation method, using highly similar auxiliary compounds. Experimental microscopic partition coefficients for triprotic molecules have only been reported before for thyroxine and its derivatives. The parabolic pH‐distribution profile of 5‐HT shows the dominance of the lipophilic non‐charged microspecies, with a log p of 0.66. However, the most lipophilic non‐charged form of 5‐HTP, with a log p of 0.31, has no significant contribution to the distribution coefficient at any pH value. Instead, the less lipophilic zwitterionic protonation isomer dominates the distribution in the pH range 2.10 – 11.11. Although the non‐charged microspecies of 5‐HTP is 151 times more lipophilic than its zwitterionic protonation isomer, the overwhelming dominance of the zwitterionic form ensures that its contribution to the overall lipophilicity exceeds 1320 times that of the non‐charged one. This fact is another counter‐example of the widespread belief that passive diffusion into lipophilic media is predominated by the non‐charged species. The lipophilicity profile of 5‐HT and 5‐HTP is depicted in terms of species‐specific lipophilicities.     

Dihydroxynaphthalene‐based mimicry of fungal melanogenesis for multifunctional coatings

Material‐independent adhesive action derived from polycatechol structures has been intensively studied due to its high applicability in surface engineering. Here, we for the first time demonstrate that a dihydroxynaphthalene‐based fungal melanin mimetic, which exhibit a catechol‐free structure, can act as a coating agent for material‐independent surface modifications on the nanoscale. This mimetic was made by using laccase to catalyse the oxidative polymerization of specifically 2,7‐dihydroxynaphthalene. Analyses of the product of this reaction, using Fourier transform infrared‐attenuated total reflectance and X‐ray photoelectron spectroscopy, bactericidal action, charge‐dependent sorption behaviour, phenol content, Zeta potential measurements and free radical scavenging activity, yielded results consistent with it containing hydroxyphenyl groups. Moreover, nuclear magnetic resonance analyses of the product revealed that C‐O coupling and C‐C coupling were the main mechanisms for its synthesis, thus clearly excluding a catechol structure in the polymerization. This product, termed poly(2,7‐DHN), was successfully deposited onto a wide variety of solid surfaces, including metals, polymeric materials, ceramics, biosurfaces and mineral complexes. The melanin‐like polymerization could be used to co‐immobilize other organic molecules, forming functional surfaces. In addition, the hydroxyphenyl group contained in the coated poly(2,7‐DHN) induced secondary metal chelation/reduction and adhesion with proteins, suggesting the potential of this poly(2,7‐DHN) layer to serve as a platform material for a variety of surface engineering applications. Moreover, the novel physicochemical properties of the poly(2,7‐DHN) illuminate its potential applications as bactericidal, radical‐scavenging and pollutant‐sorbing agents.     

Germinal‐center kinase‐like kinase co‐crystal structure reveals a swapped activation loop and C‐terminal extension

Germinal‐center kinase‐like kinase (GLK, Map4k3), a GCK‐I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation‐loop swapped dimer with more than 20 amino acids of ordered density at the carboxy‐terminus. This C‐terminal PEST region binds intermolecularly to the hydrophobic groove of the N‐terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an “active” kinase, including the salt bridge between the C‐helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P‐loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.     

Associative learning of host presence in non‐host environments influences parasitoid foraging

1. Parasitoids are known to utilise learning of herbivore‐induced plant volatiles (HIPVs) when foraging for their herbivorous host. In natural situations these hosts share food plants with other, non‐suitable herbivores (non‐hosts). Simultaneous infestation of plants by hosts and non‐hosts has been found to result in induction of HIPVs that differ from host‐infested plants. Each non‐host herbivore may have different effects on HIPVs when sharing the food plant with hosts, and thus parasitoids may learn that plants with a specific non‐host herbivore also contain the host. 2. This study investigated the adaptive nature of learning by a foraging parasitoid that had acquired oviposition experience on a plant infested with both hosts and different non‐hosts in the laboratory and in semi‐field experiments. 3. In two‐choice preference tests, the parasitoid Cotesia glomerata shifted its preference towards HIPVs of a plant–host–non‐host complex previously associated with an oviposition experience. It could, indeed, learn that the presence of its host is associated with HIPVs induced by simultaneous feeding of its host Pieris brassicae and either the non‐host caterpillar Mamestra brassicae or the non‐host aphid Myzus persicae. However, the learned preference found in the laboratory did not translate into parasitisation preferences for hosts accompanying non‐host caterpillars or aphids in a semi‐field situation. 4. This paper discusses the importance of learning in parasitoid foraging, and debates why observed learned preferences for HIPVs in the laboratory may cancel out under some field experimental conditions.     

DbpA is a region‐specific RNA helicase

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic core's grasp.     

PopW of Ralstonia solanacearum,a new two‐domain harpin targeting the plant cell wall

Harpins are extracellular glycine‐rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380‐amino‐acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non‐host), PopW induced a rapid tissue collapse via a heat‐stable but protease‐sensitive HR‐eliciting activity. PopW has an N‐terminal harpin domain (residues 1–159) and a C‐terminal pectate lyase (PL) domain (residues 160–366); its HR‐eliciting activity depends on its N‐terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB‐dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW‐deficient mutant retained the ability of wild‐type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall‐associated, hrpB‐dependent, two‐domain harpin that is conserved across the R. solanacearum species complex.