Synergistic roles of acyl‐CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism

Toxoplasma gondii relies on apicoplast‐localised FASII pathway and endoplasmic reticulum‐associated fatty acid elongation pathway for the synthesis of fatty acids, which flow through lipid metabolism mainly in the form of long‐chain acyl‐CoA (LCACoAs) esters. Functions of Toxoplasma acyl‐CoA transporters in lipid metabolism remain unclear. Here, we investigated the roles of acyl‐CoA‐binding protein (TgACBP1) and a sterol carrier protein‐2 (TgSCP2) as cytosolic acyl‐CoA transporters in lipid metabolism. The fluormetric binding assay and yeast complementation confirmed the acyl‐CoA binding activities of TgACBP1 and TgSCP2, respectively. Disruption of either TgACBP1 or TgSCP2 caused no obviously phenotypic changes, whereas double disruption resulted in defects in intracellular growth and virulence to mice. Gas chromatography coupled with mass spectrometry (GC–MS) results showed that TgACBP1 or TgSCP2 disruption alone led to decreased abundance of C18:1, whereas double disruption resulted in reduced abundance of C18:1, C22:1, and C24:1. ¹³C labelling assay combined with GC–MS showed that double disruption of TgACBP1 and TgSCP2 led to reduced synthesis rates of C18:0, C22:1, and C24:1. Furthermore, high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC‐HRMS) was used for lipidomic analysis of parasites and indicated that loss of TgACBP1 and TgSCP2 caused serious defects in production of glycerides and phospholipids. Collectively, TgACBP1 and TgSCP2 play synergistic roles in lipid metabolism in T. gondii.     

The NIN‐like protein 5 (ZmNLP5) transcription factor is involved in modulating the nitrogen response in maize

Maize exhibits marked growth and yield response to supplemental nitrogen (N). Here, we report the functional characterization of a maize NIN‐like protein ZmNLP5 as a central hub in a molecular network associated with N metabolism. Predominantly expressed and accumulated in roots and vascular tissues, ZmNLP5 was shown to rapidly respond to nitrate treatment. Under limited N supply, compared with that of wild‐type (WT) seedlings, the zmnlp5 mutant seedlings accumulated less nitrate and nitrite in the root tissues and ammonium in the shoot tissues. The zmnlp5 mutant plants accumulated less nitrogen than the WT plants in the ear leaves and seed kernels. Furthermore, the mutants carrying the transgenic ZmNLP5 cDNA fragment significantly increased the nitrate content in the root tissues compared with that of the zmnlp5 mutants. In the zmnlp5 mutant plants, loss of the ZmNLP5 function led to changes in expression for a significant number of genes involved in N signalling and metabolism. We further show that ZmNLP5 directly regulates the expression of nitrite reductase 1.1 (ZmNIR1.1) by binding to the nitrate‐responsive cis‐element at the 5′ UTR of the gene. Interestingly, a natural loss‐of‐function allele of ZmNLP5 in Mo17 conferred less N accumulation in the ear leaves and seed kernels resembling that of the zmnlp5 mutant plants. Our findings show that ZmNLP5 is involved in mediating the plant response to N in maize.     

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1‐like protein

Dynamin 1‐like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.     

Crystal structure of human WBSCR16, an RCC1‐like protein in mitochondria

WBSCR16 (Williams‐Beuren Syndrome Chromosomal Region 16) gene is located in a large deletion region of Williams‐Beuren syndrome (WBS), which is a neurodevelopmental disorder. Although the relationship between WBSCR16 and WBS remains unclear, it has been reported that WBSCR16 is a member of a functional module that regulates mitochondrial 16S rRNA abundance and intra‐mitochondrial translation. WBSCR16 has RCC1 (Regulator of Chromosome Condensation 1)‐like amino acid sequence repeats but the function of WBSCR16 appears to be different from that of other RCC1 superfamily members. Here, we demonstrate that WBSCR16 localizes to mitochondria in HeLa cells, and report the crystal structure of WBSCR16 determined to 2.0 Å resolution using multi‐wavelength anomalous diffraction. WBSCR16 adopts the seven‐bladed β‐propeller fold characteristic of RCC1‐like proteins. A comparison of the WBSCR16 structure with that of RCC1 and other RCC1‐like proteins reveals that, although many of the residues buried in the core of the β‐propeller are highly conserved, the surface residues are poorly conserved and conformationally divergent.     

Di‐2‐ethylhexyl phthalate (DEHP) exposure disturbs lipid metabolism in juvenile yellow catfish Tachysurus fulvidraco

This study was conducted to determine the mechanism by which di‐2‐ethylhexyl phthalate (DEHP) exposure influences lipid metabolism of juvenile yellow catfish Tachysurus fulvidraco. Fish were exposed to three DEHP concentrations (0, 0·1 and 0·5 mg l^?1 DEHP) for 8 weeks. Fatty acid synthase (FAS) activity significantly decreased with increasing DEHP concentrations, the highest value was in the Tween control group, whereas the lowest activities of carnitine palmitoyltransferase (CPT) and lipoprotein lipase (LPL) were in this group. The messenger (m)RNA levels of 6‐phospho‐gluconate dehydrogenase (6PGD), FAS and acetyl‐CoA carboxylase a (ACCa) significantly increased with increasing DEHP concentration, the highest values were in the 0·5 mg l^?1 DEHP group. The mRNA level of peroxisome proliferator‐activated receptor γ (PPARγ) was lower in Tween control than in fish exposed to 0·1 and 0·5 mg l^?1 DEHP. The highest mRNA level of ACCb was in the 0·1 mg l^?1 DEHP group. These results indicate that DEHP exposure can disturb lipid metabolism at the enzymatic and mRNA levels in Pelteobagrus fulvidraco.     

The GmFWL1 (FW2‐2‐like) nodulation gene encodes a plasma membrane microdomain‐associated protein

The soybean gene GmFWL1 (FW2‐2‐like1) belongs to a plant‐specific family that includes the tomato FW2‐2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2‐2‐like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain‐associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.     

The metabolic acclimation of Arabidopsis thaliana to arsenate is sensitized by the loss of mitochondrial LIPOAMIDE DEHYDROGENASE2, a key enzyme in oxidative metabolism

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild‐type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady‐state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild‐type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis.     

Mouse fat storage‐inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

Fat storage‐inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)‐localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension‐cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER‐LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER‐vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.     

Tumour necrosis factor‐α‐induced protein 8‐like 2 is a novel regulator of proliferation,migration, and invasion in human rectal adenocarcinoma cells

Tumour necrosis factor‐α‐induced protein 8‐like 2 (TIPE2) is a tumour suppressor in many types of cancer. However, the mechanism of action of TIPE2 on the growth of rectal adenocarcinoma is unknown. Our results showed that the expression levels of TIPE2 in human rectal adenocarcinoma tissues were higher than those in adjacent non‐tumour tissues. Overexpression of TIPE2 reduced the proliferation, migration, and invasion of human rectal adenocarcinoma cells and down‐regulation of TIPE2 showed reverse effects. TIPE2 overexpression increased apoptosis through down‐regulating the expression levels of Wnt3a, phospho (p)‐β‐Catenin, and p‐glycogen synthase kinase‐3β in rectal adenocarcinoma cells, however, TIPE2 knockdown exhibited reverse trends. TIPE2 overexpression decreased autophagy by reducing the expression levels of p‐Smad2, p‐Smad3, and transforming growth factor‐beta (TGF‐β) in rectal adenocarcinoma cells, however, TIPE2 knockdown showed opposite effects. Furthermore, TIPE2 overexpression reduced the growth of xenografted human rectal adenocarcinoma, whereas TIPE2 knockdown promoted the growth of rectal adenocarcinoma tumours by modulating angiogenesis. In conclusion, TIPE2 could regulate the proliferation, migration, and invasion of human rectal adenocarcinoma cells through Wnt/β‐Catenin and TGF‐β/Smad2/3 signalling pathways. TIPE2 is a potential therapeutic target for the treatment of rectal adenocarcinoma.     

Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

Characterization,expression analysis and localization pattern of toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes in grass carp Ctenopharyngodon idella

In this study, the toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes of grass carp Ctenopharyngodon idella were cloned and characterized. tlr1 and tlr2 were found to be highly expressed in immune system organs such as spleen, middle kidney and heart kidney. The expression level of tlr1 and tlr2 was found to be up‐regulated at the later stage of viral challenge process. Moreover, subcellular localization indicated that Tlr1 and Tlr2 shared similar localization pattern and both of them may locate in the plasma membrane of transfected cells.     

Crystal structure of heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding

The heart‐specific isoform of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well‐known inhibitor of PFKFB2, co‐crystallized in the 2‐kinase domains of both orthologues, occupying the fructose‐6‐phosphate binding‐site and extending into the γ‐phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ‐phosphate site by citrate proved highly consequential to the binding of co‐complexed ATP analogues. The bovine structure, which co‐crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co‐complexed with AMPPNP, which, unlike ADP, contains a γ‐phosphate. The presence of this γ‐phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding‐pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed‐back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117–124. © 2016 Wiley Periodicals, Inc.     

The ABCs of flower development: mutational analysis of AP1/FUL‐like genes in rice provides evidence for a homeotic (A)‐function in grasses

The well‐known ABC model describes the combinatorial interaction of homeotic genes in specifying floral organ identities. While the B‐ and C‐functions are highly conserved throughout flowering plants and even in gymnosperms, the A‐function, which specifies the identity of perianth organs (sepals and petals in eudicots), remains controversial. One reason for this is that in most plants that have been investigated thus far, with Arabidopsis being a remarkable exception, one does not find recessive mutants in which the identity of both types of perianth organs is affected. Here we report a comprehensive mutational analysis of all four members of the AP1/FUL‐like subfamily of MADS‐box genes in rice (Oryza sativa). We demonstrate that OsMADS14 and OsMADS15, in addition to their function of specifying meristem identity, are also required to specify palea and lodicule identities. Because these two grass‐specific organs are very likely homologous to sepals and petals of eudicots, respectively, we conclude that there is a floral homeotic (A)‐function in rice as defined previously. Together with other recent findings, our data suggest that AP1/FUL‐like genes were independently recruited to fulfil the (A)‐function in grasses and some eudicots, even though other scenarios cannot be excluded and are discussed.     

The extracellular β‐1,3‐endoglucanase EngA is involved in autolysis of Aspergillus nidulans

Aims: To elucidate the roles of the β‐1,3‐endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. Methods and Results: A β‐1,3‐endoglucanase was purified from carbon‐starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene‐expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. Conclusions: The β‐1,3‐endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall–degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. Significance and Impact of the Study: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.     

Effects of toxic β‐glucosides on carbohydrate metabolism in cotton bollworm,Helicoverpa armigera (Hübner)

The purpose of this study was to evaluate the effects of three toxic β‐glucosides, phlorizin, santonin, and amygdalin, on carbohydrate metabolism in the cotton bollworm, Helicoverpa armigera (Hübner), when diets mixed with β‐glucosides were fed to third‐instar larvae. The growth of the larvae was significantly inhibited by exposure to santonin after 96 hr but not obviously affected by phlorizin and amygdalin. The midgut trehalase activities were only 51.7%, 32%, and 42.5% of that of the control after treatment with phlorizin, santonin and amygdalin at 2 mg/ml, respectively. In the hemolymph and fat body, the amount of trehalose decreased in all cases. However, the effects of santonin on the alteration of the glycogen and glucose levels as well as the activities of glycogen phosphorylase, were different than those of the other two β‐glucosides. It appears that the three β‐glucosides have different influences on the carbohydrate metabolism of cotton bollworm.