C-terminal GFP Research Articles

NO dioxygenase- and peroxidase-like activity of Arabidopsis phytoglobin 3 and its role in Sclerotinia sclerotiorum defense

Phytoglobin 3 appears to be ubiquitous in plants, yet there has been dearth of evidence for their potent physiological functions. Previous crystallographic studies suggest a potential NO dioxygenase like activity of Arabidopsis phytoglobin 3 (AHb3). The present work examined the in vivo function of AHb3 in plant physiology and its role in biotic stress using Arabidopsis- Sclerotinia sclerotorium pathosystem. The gene was found to be ubiquitously expressed in all plant tissues, with moderately increased expression in roots. Its expression was induced upon NO, H2O2 and biotic stress. A C-terminal tagged GFP version of the wild type protein revealed its enhanced accumulation in the guard cells. AHb3-GFP was found to be partitioned majorly into the nucleus while residual amounts were present in the cytoplasm. The loss of function AHb3 mutant exhibited reduced root length and fresh weight. AHb3 knockout lines also displayed enhanced susceptibility towards the S. sclerotiorum. Interestingly, these lines displayed enhanced ROS accumulation upon pathogen challenge as suggested by DAB staining. Furthermore, enhanced/decreased NO accumulation in AHb3 knockout/overexpression lines upon treatment with multiple NO donors suggests a potent NO dioxygenase like activity for the protein. Taken together, our data indicate that AHb3 play a crucial role in regulating root length as well as in mediating defense response against S. sclerotiorum, possibly by modulating NO and ROS levels.

Co-expression with the Type 3 Secretion Chaperone CesT from Enterohemorrhagic E. coli Increases Accumulation of Recombinant Tir in Plant Chloroplasts.

Type 3 secretion systems (T3SSs) are utilized by pathogenic Escherichia coli to infect their hosts and many proteins from these systems are affected by chaperones specific to T3SS-containing bacteria. Toward developing a recombinant vaccine against enterohaemorrhagic E. coli (EHEC), we expressed recombinant T3SS and related proteins from predominant EHEC serotypes in Nicotiana chloroplasts. Nicotiana benthamiana were transiently transformed to express chloroplast-targeted Tir, NleA, and EspD from the EHEC serotype O157:H7; a fusion of EspA proteins from serotypes O157:H7 and O26:H11; and a fusion of epitopes of Tir (Tir-ep) from serotypes O157:H7, O26:H11, O45:H2, and O111:H8. C-terminal GFP reporter fusion constructs were also developed and transiently expressed to confirm subcellular localization and quantify relative expression levels in situ. Recombinant proteins were co-expressed with chaperones specific to each T3SS protein with the goal of increasing their accumulation in the chloroplast. We found that co-expression with the chloroplast-targeted chaperone CesT significantly increases accumulation of recombinant Tir when the latter is either transiently expressed in the nucleus and targeted to the chloroplast of N. benthamiana or stably expressed in transplastomic Nicotiana tabacum. CesT also helped maintain higher levels of Tir:GFP fusion protein over time both in vivo and ex vivo, indicating that the favorable effect of CesT on accumulation of Tir is not specific to a single time point or to fresh material. By contrast, T3SS chaperones CesT, CesAB, CesD, and CesD2 did not increase accumulation of NleA:GFP, EspA:GFP, or EspD:GFP, which suggests dissimilar functioning of these chaperone–substrate combinations. CesT did not increase accumulation of Tir-ep:GFP, which may be due to the absence of the CesT binding domain from this fusion protein. The fusion to GFP improved accumulation of Tir-ep relative to the unfused protein, but not for the other recombinant proteins. These results emphasize the importance of native chaperones and stabilizing fusions as potential tools for the production of higher levels of recombinant proteins in plants; and may have implications for understanding interactions between T3SS chaperones and their substrates. In particular, our findings highlight the potential of T3SS chaperones to increase accumulation of recombinant T3SS proteins in heterologous systems.

In Vivo Modeling of the Pathogenic Effect of Copper Transporter Mutations That Cause Menkes and Wilson Diseases, Motor Neuropathy, and Susceptibility to Alzheimer's Disease

Copper is an essential biometal, and several inherited diseases are directly associated with a disruption to normal copper homeostasis. The best characterized are the copper deficiency and toxicity disorders Menkes and Wilson diseases caused by mutations in the p-type Cu-ATPase genes ATP7A and ATP7B, respectively. Missense mutations in the C-terminal portion of ATP7A have also been shown to cause distal motor neuropathy, whereas polymorphisms in ATP7B are associated with increased risk of Alzheimer's disease. We have generated a single, in vivo model for studying multiple pathogenic mutations in ATP7 proteins using Drosophila melanogaster, which has a single orthologue of ATP7A and ATP7B. Four pathogenic ATP7A mutations and two ATP7B mutations were introduced into a genomic ATP7 rescue construct containing an in-frame C-terminal GFP tag. Analysis of the wild type ATP7-GFP transgene confirmed that ATP7 is expressed at the basolateral membrane of larval midgut copper cells and that the transgene can rescue a normally early lethal ATP7 deletion allele to adulthood. Analysis of the gATP7-GFP transgenes containing pathogenic mutations showed that the function of ATP7 was affected, to varying degrees, by all six of the mutations investigated in this study. Of particular interest, the ATP7BK832R Alzheimer's disease susceptibility allele was found, for the first time, to be a loss of function allele. This in vivo system allows us to assess the severity of individual ATP7A/B mutations in an invariant genetic background and has the potential to be used to screen for therapeutic compounds able to restore function to faulty copper transport proteins.

De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions.

Cytochromes P450 (CYP) are attractive enzyme targets in biotechnology as they catalyze stereospecific C-hydroxylations of complex core skeletons at positions that typically are difficult to access by chemical synthesis. Membrane bound CYPs are involved in nearly all plant pathways leading to the formation of high-value compounds. In the present study, we systematically maximize the heterologous expression of six different plant-derived CYP genes in Escherichia coli, using a workflow based on C-terminal fusions to the green fluorescent protein. The six genes can be over-expressed in both K- and B-type E. coli strains using standard growth media. Furthermore, sequences encoding a small synthetic peptide and a small bacterial membrane anchor markedly enhance the expression of all six genes. For one of the CYPs, the length of the linker region between the predicted N-terminal transmembrane segment and the soluble domain is modified, in order to verify the importance of this region for enzymatic activity. The work describes how membrane bound CYPs are optimally produced in E. coli and thus adds this plant multi-membered key enzyme family to the toolbox for bacterial cell factory design.

An expression tag toolbox for microbial production of membrane bound plant cytochromes P450.

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here, we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalyzed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than twofold. The developed toolbox serves as a platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals. Biotechnol. Bioeng. 2017;114: 751-760. © 2016 Wiley Periodicals, Inc.

Novel Hydrophobin Fusion Tags for Plant-Produced Fusion Proteins.

Hydrophobin fusion technology has been applied in the expression of several recombinant proteins in plants. Until now, the technology has relied exclusively on the Trichoderma reesei hydrophobin HFBI. We screened eight novel hydrophobin tags, T. reesei HFBII, HFBIII, HFBIV, HFBV, HFBVI and Fusarium verticillioides derived HYD3, HYD4 and HYD5, for production of fusion proteins in plants and purification by two-phase separation. To study the properties of the hydrophobins, we used N-terminal and C-terminal GFP as a fusion partner. Transient expression of the hydrophobin fusions in Nicotiana benthamiana revealed large variability in accumulation levels, which was also reflected in formation of protein bodies. In two-phase separations, only HFBII and HFBIV were able to concentrate GFP into the surfactant phase from a plant extract. The separation efficiency of both tags was comparable to HFBI. When the accumulation was tested side by side, HFBII-GFP gave a better yield than HFBI-GFP, while the yield of HFBIV-GFP remained lower. Thus we present here two alternatives for HFBI as functional fusion tags for plant-based protein production and first step purification.

Recombinant Adeno Associated Viral (AAV) vector type 9 delivery of Ex1-Q138-mutant huntingtin in the rat striatum as a short-time model for in vivo studies in drug discovery

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by dyskinesia, cognitive impairment and emotional disturbances, presenting progressive neurodegeneration in the striatum and intracellular mutant Huntingtin (mHTT) aggregates in various areas of the brain. Recombinant Adeno Associated Viral (rAAV) vectors have been successfully used to transfer foreign genes to the brain of adult animals. In the present study we report a novel in vivo rat HD model obtained by stereotaxic injection of rAAV serotype2/9 containing Exon1-Q138 mHTT (Q138) and Exon1-Q17 wild type HTT (Q17; control), respectively in the right and in the left striatum, and expressed as C-terminal GFP fusions to facilitate detection of infected cells and aggregate production. Immunohistochemical analysis of brain slices from animals sacrificed twenty-one days after viral infection showed that Q138 injection resulted in robust formation of GFP-positive aggregates in the striatum, increased GFAP and microglial activation and neurodegeneration, with little evidence of any of these events in contralateral tissue infected with wild type (Q17) expressing construct. Differences in the relative metabolite concentrations (N-Acetyl Aspartate/Creatine and Myo-Inositol/Creatine) were observed by H1 MR Spectroscopy. By quantitative RT-PCR we also demonstrated that mHTT induced changes in the expression of genes previously shown to be altered in other rodent HD models. Importantly, administration of reference compounds previously shown to ameliorate the aggregation and neurodegeneration phenotypes in preclinical HD models was demonstrated to revert the mutant HTT-dependent effects in our model. In conclusion, the AAV2/9-Q138/Q17 exon 1 HTT stereotaxic injection represents a useful first-line in vivo preclinical model for studying the biology of mutant HTT exon 1 in the striatum and to provide early evidence of efficacy of therapeutic approaches.

Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana.

A subset of genes in Arabidopsis thaliana is known to be up-regulated in response to a wide range of different environmental stress factors. However, not all of these genes are characterized as yet with respect to their functions. In this study, we used transgenic knockout, overexpression and reporter gene approaches to try to elucidate the biological roles of five unknown multiple-stress responsive genes in Arabidopsis. The selected genes have the following locus identifiers: At1g18740, At1g74450, At4g27652, At4g29780 and At5g12010. Firstly, T-DNA insertion knockout lines were identified for each locus and screened for altered phenotypes. None of the lines were found to be visually different from wildtype Col-0. Secondly, 35S-driven overexpression lines were generated for each open reading frame. Analysis of these transgenic lines showed altered phenotypes for lines overexpressing the At1g74450 ORF. Plants overexpressing the multiple-stress responsive gene At1g74450 are stunted in height and have reduced male fertility. Alexander staining of anthers from flowers at developmental stage 12–13 showed either an absence or a reduction in viable pollen compared to wildtype Col-0 and At1g74450 knockout lines. Interestingly, the effects of stress on crop productivity are most severe at developmental stages such as male gametophyte development. However, the molecular factors and regulatory networks underlying environmental stress-induced male gametophytic alterations are still largely unknown. Our results indicate that the At1g74450 gene provides a potential link between multiple environmental stresses, plant height and pollen development. In addition, ruthenium red staining analysis showed that At1g74450 may affect the composition of the inner seed coat mucilage layer. Finally, C-terminal GFP fusion proteins for At1g74450 were shown to localise to the cytosol.

Abstract 3274: Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties

Abstract Background: Epidermal Growth Factor Receptor (EGFR) is a biologic target for cancer therapy. Clinical response to EGFR inhibitors is variable. Recently, EGFR mutations were found to predict response to anti-EGFR therapy. Since a few children with Neuroblastoma (NB) benefited from EGFR inhibitors in clinical trials, we screened for EGFR mutations in primary NB. Interestingly, we identified the expression of both EGFRvIII and a novel mutation, EGFRΔ768, in these tumors. In this study, we characterized the EGFRΔ768 mutation by comparing its biological and biochemical effect with that of wild type (WT)-EGFR and EGFRvIII. Hypothesis: EGFRΔ768 expression confers an aggressive cancer phenotype in NB cells Materials and Methods: We obtained 62 Snap Frozen primary neuroblastic tumors from the Columbus Bipathology Repository Center and 80 NB RNA from the Children Oncology Group (COG). Primary NB were prepared for RT-PCR and sequencing analyses using EGFR exon 1 & 8 primers. Five NB cell lines were obtained and confirmed by STR profiling. The EGFRΔ768 and EGFRvIII deletions were subcloned into a WT-EGFR expression vector with a C-terminal GFP tag. Next, stable 3T3 and NB clones expressing WT and mutant EGFR were generated. The clones were visualized under fluorescence microscopy. Invasion and XTT assays were performed with the stable clones. Responses to Erlotinib from 0.01 - 1 ng/ul and Etoposide from 0.5 - 2 uM were compared among the clones by XTT and western analyses. EGFR phosphorylation and downstream signaling pathways were examined by Western Blot and phosphokinase array. Results: We previously reported that 32% (20/62) of the Columbus neuroblastic tumors expressed EGFR extracellular deletion mutants, EGFRΔ768 (an in-frame deletion from nucleotides 102 of exon 2 to 869 of exon 7) and EGFRvIII (Δ801). In the 80 COG NB RNA, 7 expressed EGFRΔ768, 7 expressed EGFRvIII and 1 expressed both. EGFRΔ768 expression was also detected in the NB cell line, BE2M17. This novel mutant localized to the cell surface and enhanced the invasion and proliferation of NB cells in vitro when compared to control GFP+ cells. Its expression also confers sensitivity to Erlotinib, but the biochemical inhibition of Erlotinib on EGFRΔ768 is significantly less effective than that on activated WT-EGFR from 0.01 - 0.1 ng/ul. EGFRΔ768+ cells were also significantly more resistant to Etoposide than the GFP and WT-EGFR+ cells. Although both EGFRΔ768 and EGFRvIII autophosphorylated without ligand, the cellular proliferation rate and baseline autophosphorylation of EGFRΔ768+ cells were significantly higher than those of EGFRvIII+ cells. Finally, we found that EGFRΔ768 did not activate downstream signaling pathways of WT-EGFR such as MAPK or STAT. It has a different tyrosine phosphorylation profile than WT-EGFR. Conclusion: EGFRΔ768 confers aggressive cancer cell behaviors and has distinct biological as well as biochemical properties. Citation Format: James Keller, Kristen A. VanHeyst, Anjaruwee S. Nimnual, Mathew Varghese, Michael J. Hayman, Edward L. Chan. Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3274. doi:10.1158/1538-7445.AM2015-3274

AtPGL3 is an Arabidopsis BURP domain protein that is localized to the cell wall and promotes cell enlargement.

The BURP domain is a plant-specific domain that has been identified in secretory proteins, and some of these are involved in cell wall modification. The tomato polygalacturonase I complex involved in pectin degradation in ripening fruits has a non-catalytic subunit that has a BURP domain. This protein is called polygalacturonase 1 beta (PG1β) and the Arabidopsis genome encodes three proteins that exhibit strong amino acid similarities with PG1β? We generated Arabidopsis lines in which expression levels of AtPGLs are altered in order to investigate the biological roles of the Arabidopsis PG1β-like proteins (AtPGLs). Among the three AtPGLs (AtPGL1-3), AtPGL3 exhibited the highest transcriptional activity throughout all developmental stages. AtPGL triple mutant plants have smaller rosette leaves than those of wild type plants because the leaf cells are smaller in the mutant plants. Interestingly, when we overexpressed AtPGL3 using a 35S promoter, leaf cells in transgenic plants grew larger than those of the wild type. A C-terminal GFP fusion protein of AtPGL3 complemented phenotypes of the triple mutant plants and it localized to the cell wall. A truncated AtPGL3-GFP fusion protein lacking the BURP domain failed to rescue the mutant phenotypes even though the GFP protein was targeted to the cell wall, indicating that the BURP domain is required for the protein's effect on cell expansion. Quantitative RT-PCR and immunoblot analyses indicated that the α-expansin 6 gene is up-regulated in the overexpressor plants. Taken together, these results indicate that AtPGL3 is an apoplastic BURP domain protein playing a role in cell expansion.

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

Mechanosensitivity of TRPC6 Ion Channels

The transient receptor potential (TRP) ion channel family is a diverse group of channels gated by various physical and chemical stimuli. One of the members, transient receptor potential canonical-6 (TRPC6), is a calcium permeable cation channel (PCa/PNa ∼ 6) located in the T-tubules of ventricular cardiomyocytes. Abnormal TRPC6 activity has been implicated in cardiac hypertrophy, a maladaptive process likely triggered by increased mechanical stress. As a result, it is important to elucidate the gating mechanism of TRPC6. In addition to activation by diacylglycerol (DAG) TRPC6 has been reported to be also activated by mechanical force, though this has not been supported by other studies. In this study, we investigated the mechanosensitivity of TRPC6 overexpressed in HEK293 cells using the patch clamp technique. Three TRPC6 constructs were utilised: N- terminal fusion GFP, C-terminal fusion GFP and non-GFP fusion TRPC6. We have successfully transfected these constructs into HEK293 cells with a 60% transfection efficiency rate using Lipofectamine® 3000 reagent. Confocal images of C-GFP TRPC6 demonstrated that the channel is localised in the plasma membrane. However, N-GFP and non-GFP TRPC6 both distributed uniformly throughout the cells suggestive of impaired trafficking. In our patch clamp experiments we recorded TRPC6 single channel activity in cell-attached and whole-cell configurations. By applying negative pressure (suction) of −50 mmHg to the patch pipette we were able to record single channel activity of TRPC6 channels. Moreover, the channels were spontaneously active without application of negative pressure to the patch pipette suggesting that the membrane tension resulting from the gigaohm seal formation was sufficient to activate TRPC6. These results demonstrate that TRPC6 expressed in the plasma membrane of HEK293 cells is mechanosensitive. Future studies will focus on purifying and incorporating the TRPC6 channel protein into liposomes to establish if TRPC6 is intrinsically mechanosensitive.

Design and characterization of a modular membrane protein anchor to functionalize the moss Physcomitrella patens with extracellular catalytic and/or binding activities.

Heterologous enzymes and binding proteins were secreted by the moss Physcomitrella patens or anchored extracellularly on its cell membrane in order to functionalize the apoplast as a biochemical reaction compartment. This modular membrane anchoring system utilizes the signal peptide and the transmembrane segment of the somatic embryogenesis receptor-like kinase (SERK), which were identified in a comprehensive bioinformatic analysis of the P. patens genome. By fusing the soluble enzyme NanoLuc luciferase to the signal peptide, its secretion capability was confirmed in vivo. The membrane localization of hybrid proteins comprising the SERK signal peptide, NanoLuc or other functional modules, the SERK transmembrane anchor, and a C-terminal GFP reporter was demonstrated using fluorescence microscopy as well as site-specific proteolytic release of the extracellular enzyme domain. Our membrane anchoring system enables the expression of various functional proteins in the apoplast of P. patens, empowering this photoautotrophic organism for biotechnological applications.

Golgi-located NTPDase1 of Leishmania major is required for lipophosphoglycan elongation and normal lesion development whereas secreted NTPDase2 is dispensable for virulence.

Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.

The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase.

The exosome is a conserved multi-subunit ribonuclease complex that functions in 3' end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N-terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N-terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C-terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6-Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.

Characterization of New Polyol/H+ Symporters in Debaryomyces hansenii

Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H+ (and sugar/H+) symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H+ symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H+ symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H+ 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H+ 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.

Spectroscopic System for Characterization of Human β-Cardiac Myosin

We designed a system to examine the structural properties of human β-cardiac myosin using fluorescence spectroscopy. Expression of human β-cardiac myosin subfragment 1 (M2B-S1) was achieved through the generation of recombinant adenovirus with subsequent infection in the C2C12 embryonic muscle cell line. A tetracysteine site, which enables site-specific labeling of the fluorescein biarscenical hairpin-binding dye (FlAsH), was introduced in the upper 50 kDa (U50) region (residues 316-321) corresponding to the previously utilized site in myosin V. Key conformational changes in the myosin motor, including those associated with nucleotide and actin binding, can be examined by fluorescence resonance energy transfer (FRET) using M2B-S1 FlAsH and fluorescent nucleotides or IAEDANs-labeled actin. We demonstrate the enzymatic properties of M2B-S1 FlAsH are similar to unlabeled M2B-S1, and both are capable of moving actin filaments in the in vitro motility assay. We utilized FRET between M2B-S1 FlAsH and Cy3ATP to examine structural changes in the U50 domain. The kinetics of structural changes were examined during the ATP-binding and actin-activated product release steps. We also designed a system to study structural changes in the force-generating lever arm using FlAsH labeling at the N-terminus of a human β-cardiac myosin construct containing a C-terminal GFP (M2B-GFP). The spectroscopic approach can be used as a tool to study the effects of hypertrophic cardiomyopathy (HCM) mutations on myosin structure or to study the impact of drugs that alter myosin motor activity.

Development of Molecular Shuttle Regulated by External Stimulation Utilizing Kinesin ATP Driven Motor

Kinesin is known as a dimeric motor protein, which carries cellular cargoes along microtubules by hydrolyzing ATP. Its structure and the molecular mechanism of energy transduction to move along microtubule are well studied. The kinesin has many possibility of application to the molecular machines. Previously, we introduced photochromic molecules into the functional key region of the kinesin as a photoswitch and tried to control the function of kinesin by ultraviolet (UV) and visible (VIS) light irradiations. The kinesin mutant S275C modified with thiol reactive azobenzene derivative exhibited photocontrolled ATPase activity correlating to photo isomerization. We have also demonstrated that kinesin fused with CaM at the C-terminal binds reversibly to M13-Qdots in a calcium dependent manner. As mentioned above, kinesin works as a nanodevice to be a component of bionanomachines. In this study, we tried to prepare the molecular shuttles regulated by external stimulation utilizing kinesin and other functional proteins. We designed the two chimeric kinesins. One is the kinesin motor domain (K355) fused with calmodulin (CaM) and the other is K355 fused with calmodulin target peptide M13. The chimeras were expressed by E. coli. and purified by Co-Chelate column. These kinesin chimeras showed normal ATPase activities. K355-CaM bound to K355-M13 in a calcium dependent manner and formed dimer. The dimer composed of the two chimeras induced exhibited motor activity to induce microtubule gliding. Moreover, we also tried to prepare the chimeric kinesins, K355-CaM-split GFP C-terminal domain and K355-split GFP N-terminal domain to be dimerized and fluorescently labelled spontaneously.

Class III Myosin Motor Activity Correlates with Localization in Actin Protrusions

Class III myosins (myo3A and myo3B) contain a N-terminal kinase domain, central motor domain, and an isoform specific C-terminal tail. Myo3A contains a longer tail and has an actin-binding motif at the C-terminus that is not present in the shorter tail of myo3B. Modifications to the myo3A gene result in late onset human deafness (DFNB30), suggesting myo3B may be able to compensate for myo3A only early in life. Thus, it is critical to understand differences in the motor properties of myo3A and myo3B. We investigated the in vitro motility of myo3A and myo3B, each with the kinase domain removed and containing two IQ domains and a C-terminal GFP fusion (myo3AΔK.GFP and myo3BΔK.GFP). The sliding velocity measured in the in vitro motility assay was ∼20% faster in myo3AΔK.GFP (76 nm/sec) compared to myo3BΔK.GFP (62 nm/sec). The sliding velocity correlated well with the maximum actin-activated ATPase activity (kcat = 1.0 sec−1 and 0.7 sec−1, respectively). Interestingly, myo3B.ΔK.GFP contained a 25-fold weaker actin affinity compared to myo3A.ΔK.GFP as assessed by the actin-dependence of the ATPase activity (KATPase = 3 and 76 µM, respectively). We transfected N-terminally GFP tagged myo3A.ΔK containing the full length tail domain into COS7 cells and examined the efficiency of localizing to the filopodia tips by examining the tip to cell body ratio. Since the tail actin binding motif is required for localization to filopodia tips, we generated a chimera that contained the myo3B motor and the actin binding motif of the myo3A tail. The tip localization efficiency was approximately 17% (p<0.05) reduced in myo3B compared to myo3A. Thus myo3B is a slightly slower motor with a weaker actin affinity compared to myo3A, which may explain why it is unable to compensate for the loss of myo3A in DFNB30.

The dual targeting ability of type II NAD(P)H dehydrogenases arose early in land plant evolution

BackgroundType II NAD(PH) dehydrogenases are located on the inner mitochondrial membrane of plants, fungi, protists and some primitive animals. However, recent observations have been made which identify several Arabidopsis type II dehydrogenases as dual targeted proteins. Targeting either mitochondria and peroxisomes or mitochondria and chloroplasts.ResultsMembers of the ND protein family were identified in various plant species. Phylogenetic analyses and subcellular targeting predictions were carried out for all proteins. All ND proteins from three model plant species Arabidopsis, rice and Physcomitrella were cloned as N- and C-terminal GFP fusions and subcellular localisations were determined. Dual targeting of plant type II dehydrogenases was observed to have evolved early in plant evolution and to be widespread throughout different plant species. In all three species tested dual targeting to both mitochondria and peroxisomes was found for at least one NDA and NDB type protein. In addition two NDB type proteins from Physcomitrella were also found to target chloroplasts. The dual targeting of NDC type proteins was found to have evolved later in plant evolution.ConclusionsThe functions of type II dehydrogenases within plant cells will have to be re-evaluated in light of this newly identified subcellular targeting information.