Publications by authors named "Meredith Morris"

31 Publications

Regulation of Fructose 1,6-Bisphosphatase in Procyclic Form .

Pathogens 2021 May 18;10(5). Epub 2021 May 18.

Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Life Sciences Facility, 190 Collings Ave., Clemson, SC 29634, USA.

Glycolysis is well described in , while the importance of gluconeogenesis and one of the key enzymes in that pathway, fructose 1,6-bisphosphatase, is less understood. Using a sensitive and specific assay for FBPase, we demonstrate that FBPase activity in insect stage, procyclic form (PF), parasite changes with parasite cell line, extracellular glucose levels, and cell density. FBPase activity in log phase PF 2913 cells was highest in high glucose conditions, where gluconeogenesis is expected to be inactive, and was undetectable in low glucose, where gluconeogenesis is predicted to be active. This unexpected relationship between FBPase activity and extracellular glucose levels suggests that FBPase may not be exclusively involved in gluconeogenesis and may play an additional role in parasite metabolism. In stationary phase cells, the relationship between FBPase activity and extracellular glucose levels was reversed. Furthermore, we found that monomorphic PF 2913 cells had significantly higher FBPase levels than pleomorphic PF AnTat1.1 cells where the activity was undetectable except when cells were grown in standard SDM79 media, which is glucose-rich and commonly used to grow PF trypanosomes in vitro. Finally, we observed several conditions where FBPase activity changed while protein levels did not, suggesting that the enzyme may be regulated via post-translational modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pathogens10050617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157246PMC
May 2021

Glycosome heterogeneity in kinetoplastids.

Biochem Soc Trans 2021 02;49(1):29-39

Department of Genetics and Biochemistry, Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC 29634, U.S.A.

Kinetoplastid parasites have essential organelles called glycosomes that are analogous to peroxisomes present in other eukaryotes. While many of the processes that regulate glycosomes are conserved, there are several unique aspects of their biology that are divergent from other systems and may be leveraged as therapeutic targets for the treatment of kinetoplastid diseases. Glycosomes are heterogeneous organelles that likely exist as sub-populations with different protein composition and function in a given cell, between individual cells, and between species. However, the limitations posed by the small size of these organelles makes the study of this heterogeneity difficult. Recent advances in the analysis of small vesicles by flow-cytometry provide an opportunity to overcome these limitations. In this review, we describe studies that document the diverse nature of glycosomes and propose an approach to using flow cytometry and organelle sorting to study the diverse composition and function of these organelles. Because the cellular machinery that regulates glycosome protein import and biogenesis is likely to contribute, at least in part, to glycosome heterogeneity we highlight some ways in which the glycosome protein import machinery differs from that of peroxisomes in other eukaryotes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BST20190517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925000PMC
February 2021

Trypanosoma brucei Pex13.2 Is an Accessory Peroxin That Functions in the Import of Peroxisome Targeting Sequence Type 2 Proteins and Localizes to Subdomains of the Glycosome.

mSphere 2020 02 19;5(1). Epub 2020 Feb 19.

Eukaryotic Innovations Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA

Kinetoplastid parasites, including , , and , harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous. causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. has glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSphere.00744-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031615PMC
February 2020

A Microfluidic-Based Microscopy Platform for Continuous Interrogation of Trypanosoma brucei during Environmental Perturbation.

Biochemistry 2019 02 29;58(7):875-882. Epub 2019 Jan 29.

Department of Chemistry , Clemson University , Clemson , South Carolina 29634 , United States.

The African trypanosome, Trypanosoma brucei, is the causative agent of human African trypanosomiasis (HAT). African trypanosomes are extracellular parasites that possess a single flagellum that imparts a high degree of motility to the microorganisms. In addition, African trypanosomes show significant metabolic and structural adaptation to environmental conditions. Analysis of the ways that environmental cues affect these organisms generally requires rapid perfusion experiments in combination with single-cell imaging, which are difficult to apply under conditions of rapid motion. Microfluidic devices have been used previously as a strategy for trapping small motile cells in a variety of organisms, including trypanosomes; however, in the past, such devices required individual fabrication in a cleanroom, limiting their application. Here we demonstrate that a commercial microfluidic device, typically used for bacterial trapping, can trap bloodstream and procyclic form trypanosomes, allowing for rapid buffer exchange via perfusion. As a result, time-lapse single-cell microscopy images of these highly motile parasites were acquired during environmental variations. Using these devices, we have been able to perform and analyze perfusion-based single-cell tracking experiments of the responses of the parasite to changes in glucose availability, which is a major step in resolving the mechanisms of adaptation of kinetoplasts to their individual biological niches; we demonstrate utility of this tool for making measurements of procyclic form trypanosome intracellular glucose levels as a function of changes in extracellular glucose concentrations. These experiments demonstrate that cytosolic glucose equilibrates with external conditions as fast as, or faster than, the rate of solution exchange in the instrument.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.8b01269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463508PMC
February 2019

A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites.

PLoS Negl Trop Dis 2018 05 31;12(5):e0006523. Epub 2018 May 31.

Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, United States of America.

The bloodstream lifecycle stage of the kinetoplastid parasite Trypanosoma brucei relies solely on glucose metabolism for ATP production, which occurs in peroxisome-like organelles (glycosomes). Many studies have been conducted on glucose uptake and metabolism, but none thus far have been able to monitor changes in cellular and organellar glucose concentration in live parasites. We have developed a non-destructive technique for monitoring changes in cytosolic and glycosomal glucose levels in T. brucei using a fluorescent protein biosensor (FLII12Pglu-700μδ6) in combination with flow cytometry. T. brucei parasites harboring the biosensor allowed for observation of cytosolic glucose levels. Appending a type 1 peroxisomal targeting sequence caused biosensors to localize to glycosomes, which enabled observation of glycosomal glucose levels. Using this approach, we investigated cytosolic and glycosomal glucose levels in response to changes in external glucose or 2-deoxyglucose concentration. These data show that procyclic form and bloodstream form parasites maintain different glucose concentrations in their cytosol and glycosomes. In procyclic form parasites, the cytosol and glycosomes maintain indistinguishable glucose levels (3.4 ± 0.4mM and 3.4 ± 0.5mM glucose respectively) at a 6.25mM external glucose concentration. In contrast, bloodstream form parasites maintain glycosomal glucose levels that are ~1.8-fold higher than the surrounding cytosol, equating to 1.9 ± 0.6mM in cytosol and 3.5 ± 0.5mM in glycosomes. While the mechanisms of glucose transport operating in the glycosomes of bloodstream form T. brucei remain unresolved, the methods described here will provide a means to begin to dissect the cellular machinery required for subcellular distribution of this critical hexose.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0006523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997345PMC
May 2018

FRET Flow Cytometry-Based High Throughput Screening Assay To Identify Disrupters of Glucose Levels in Trypanosoma brucei.

ACS Infect Dis 2018 07 14;4(7):1058-1066. Epub 2018 May 14.

Department of Chemistry and Biochemistry , Brigham Young University , Provo , Utah 84601 , United States.

Trypanosoma brucei, which causes human African typanosomiasis (HAT), derives cellular ATP from glucose metabolism while in the mammalian host. Targeting glucose uptake or regulation in the parasite has been proposed as a potential therapeutic strategy. However, few methods have been described to identify and characterize potential inhibitors of glucose uptake and regulation. Here, we report development of a screening assay that identifies small molecule disrupters of glucose levels in the cytosol and glycosomes. Using an endogenously expressed fluorescent protein glucose sensor expressed in cytosol or glycosomes, we monitored intracellular glucose depletion in the different cellular compartments. Two glucose level disrupters were identified, one of which only exhibited inhibition of glycosomal glucose and did not affect cytosolic levels. In addition to inhibiting glucose uptake with relatively high potency (EC = 700 nM), the compound also showed modest bloodstream form parasite killing activity. Expanding this assay will allow for identification of candidate compounds that disrupt parasite glucose metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.8b00058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350775PMC
July 2018

Glycosome biogenesis in trypanosomes and the de novo dilemma.

PLoS Negl Trop Dis 2017 04 20;11(4):e0005333. Epub 2017 Apr 20.

Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, United States of America.

Trypanosomatid parasites, including Trypanosoma and Leishmania, are the causative agents of lethal diseases threatening millions of people around the world. These organisms compartmentalize glycolysis in essential, specialized peroxisomes called glycosomes. Peroxisome proliferation can occur through growth and division of existing organelles and de novo biogenesis from the endoplasmic reticulum. The level that each pathway contributes is debated. Current evidence supports the concerted contribution of both mechanisms in an equilibrium that can vary depending on environmental conditions and metabolic requirements of the cell. Homologs of a number of peroxins, the proteins involved in peroxisome biogenesis and matrix protein import, have been identified in T. brucei. Based on these findings, it is widely accepted that glycosomes proliferate through growth and division of existing organelles; however, to our knowledge, a de novo mechanism of biogenesis has not been directly demonstrated. Here, we review recent findings that provide support for the existence of an endoplasmic reticulum (ER)-derived de novo pathway of glycosome biogenesis in T. brucei. Two studies recently identified PEX13.1, a peroxin involved in matrix protein import, in the ER of procyclic form T. brucei. In other eukaryotes, peroxins including PEX13 have been found in the ER of cells undergoing de novo biogenesis of peroxisomes. In addition, PEX16 and PEX19 have been characterized in T. brucei, both of which are important for de novo biogenesis in other eukaryotes. Because glycosomes are rapidly remodeled via autophagy during life cycle differentiation, de novo biogenesis could provide a method of restoring glycosome populations following turnover. Together, the findings we summarize provide support for the hypothesis that glycosome proliferation occurs through growth and division of pre-existing organelles and de novo biogenesis of new organelles from the ER and that the level each mechanism contributes is influenced by glucose availability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0005333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398534PMC
April 2017

pH regulation in glycosomes of procyclic form .

J Biol Chem 2017 05 27;292(19):7795-7805. Epub 2017 Mar 27.

the Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na/H exchangers are required for glycosomal pH regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M117.784173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427261PMC
May 2017

Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum.

J Eukaryot Microbiol 2017 01 18;64(1):97-105. Epub 2016 Jul 18.

Department of Genetics and Biochemistry, Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, South Carolina, 29634.

Trypanosoma brucei is the causative agent of diseases that affect 30,000-50,000 people annually. Trypanosoma brucei harbors unique organelles named glycosomes that are essential to parasite survival, which requires growth under fluctuating environmental conditions. The mechanisms that govern the biogenesis of these organelles are poorly understood. Glycosomes are evolutionarily related to peroxisomes, which can proliferate de novo from the endoplasmic reticulum or through the growth and division of existing organelles depending on the organism and environmental conditions. The effect of environment on glycosome biogenesis is unknown. Here, we demonstrate that the glycosome membrane protein, TbPex13.1, is localized to glycosomes when cells are cultured under high glucose conditions and to the endoplasmic reticulum in low glucose conditions. This localization in low glucose was dependent on the presence of a C-terminal tripeptide sequence. Our findings suggest that glycosome biogenesis is influenced by extracellular glucose levels and adds to the growing body of evidence that de novo glycosome biogenesis occurs in trypanosomes. Because the movement of peroxisomal membrane proteins is a hallmark of ER-dependent peroxisome biogenesis, TbPex13.1 may be a useful marker for the study such processes in trypanosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jeu.12343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215699PMC
January 2017

Using fluorescent proteins to monitor glycosome dynamics in the African trypanosome.

J Vis Exp 2014 Aug 19(90):e51647. Epub 2014 Aug 19.

Department of Genetics and Biochemistry, Clemson University Eukaryotic Pathogens Innovation Center;

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions. Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses. To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/51647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827976PMC
August 2014

Environmentally regulated glycosome protein composition in the African trypanosome.

Eukaryot Cell 2013 Aug 24;12(8):1072-9. Epub 2013 May 24.

Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA.

Trypanosomes compartmentalize many metabolic enzymes in glycosomes, peroxisome-related microbodies that are essential to parasite survival. While it is understood that these dynamic organelles undergo profound changes in protein composition throughout life cycle differentiation, the adaptations that occur in response to changes in environmental conditions are less appreciated. We have adopted a fluorescent-organelle reporter system in procyclic Trypanosoma brucei by expressing a fluorescent protein (FP) fused to a glycosomal targeting sequence (peroxisome-targeting sequence 2 [PTS2]). In these cell lines, PTS2-FP is localized within import-competent glycosomes, and organelle composition can be analyzed by microscopy and flow cytometry. Using this reporter system, we have characterized parasite populations that differ in their glycosome composition. In glucose-rich medium, two parasite populations are observed; one population harbors glycosomes bearing the full repertoire of glycosome proteins, while the other parasite population contains glycosomes that lack the usual glycosome-resident proteins but do contain the glycosome membrane protein TbPEX11. Interestingly, these cells lack TbPEX13, a protein essential for the import of proteins into the glycosome. This bimodal distribution is lost in low-glucose medium. Furthermore, we have demonstrated that changes in environmental conditions trigger changes in glycosome protein composition. These findings demonstrate a level of procyclic glycosome diversity heretofore unappreciated and offer a system by which glycosome dynamics can be studied in live cells. This work adds to our growing understanding of how the regulation of glycosome composition relates to environmental sensing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00086-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754536PMC
August 2013

Peptide-targeted delivery of a pH sensor for quantitative measurements of intraglycosomal pH in live Trypanosoma brucei.

Biochemistry 2013 May 17;52(21):3629-37. Epub 2013 May 17.

Department of Chemistry and ‡Department of Genetics and Biochemistry, Clemson University , Clemson, South Carolina 29634, United States.

Studies of dynamic changes in organelles of protozoan parasite Trypanosoma brucei have been limited, in part because of the difficulty of targeting analytical probes to specific subcellular compartments. Here we demonstrate application of a ratiometric probe for pH quantification in T. brucei glycosomes. The probe consists of a peptide encoding the peroxisomal targeting sequence (F-PTS1, acetyl-CKGGAKL) coupled to fluorescein, which responds to pH. When incubated with living parasites, the probe is internalized within vesicular structures that colocalize with a glycosomal marker. Inhibition of uptake of F-PTS1 at 4 °C and pulse-chase colocalization with fluorescent dextran suggested that the probe is initially taken up by non-receptor-mediated endocytosis but is subsequently transported separately from dextran and localized within glycosomes, prior to the final fusion of labeled glycosomes and lysosomes as part of glycosomal turnover. Intraorganellar measurements and pH calibration with F-PTS1 in T. brucei glycosomes indicate that the resting glycosomal pH under physiological conditions is 7.4 ± 0.2. However, incubation in glucose-depleted buffer triggered mild acidification of the glycosome over a period of 20 min, with a final observed pH of 6.8 ± 0.3. This glycosomal acidification was reversed by reintroduction of glucose. Coupling of ratiometric fluorescent sensors and reporters to PTS peptides offers an invaluable tool for monitoring in situ glycosomal response(s) to changing environmental conditions and could be applied to additional kinetoplastid parasites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi400029mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507074PMC
May 2013

Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay.

J Biomol Screen 2013 Jul 11;18(6):714-25. Epub 2013 Mar 11.

Boston Children’s Hospital, Harvard Medical School, Vascular Biology Program, Department of Surgery, Karp 11, 300 Longwood Ave, Boston, MA 02115, USA.

Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z' value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057113478655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859190PMC
July 2013

Extra-glycosomal localisation of Trypanosoma brucei hexokinase 2.

Int J Parasitol 2012 Apr;42(4):401-9

Department of Genetics and Biochemistry, Clemson University, Clemson, SC 39634, USA.

The majority of the glycolytic enzymes in the African trypanosome are compartmentalised within peroxisome-like organelles, the glycosomes. Polypeptides harbouring peroxisomal targeting sequences (PTS type 1 or 2) are targeted to these organelles. This targeting is essential to parasite viability, as compartmentalisation of glycolytic enzymes prevents unregulated ATP-dependent phosphorylation of intermediate metabolites. Here, we report the surprising extra-glycosomal localisation of a PTS-2 bearing trypanosomal hexokinase, TbHK2. In bloodstream form parasites, the protein localises to both glycosomes and to the flagellum. Evidence for this includes fractionation and immunofluorescence studies using antisera generated against the authentic protein as well as detection of epitope-tagged recombinant versions of the protein. In the insect stage parasite, distribution is different, with the polypeptide localised to glycosomes and proximal to the basal bodies. The function of the extra-glycosomal protein remains unclear. While its association with the basal body suggests that it may have a role in locomotion in the insect stage parasite, no detectable defect in directional motility or velocity of cell movement were observed for TbHK2-deficient cells, suggesting that the protein may have a different function in the cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpara.2012.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710734PMC
April 2012

Co-located collaborative visual analytics around a tabletop display.

IEEE Trans Vis Comput Graph 2012 May;18(5):689-702

Université Paris-Sud, Equipe Aviz Bat 490, Saclay, Orsay 91405, France.

Co-located collaboration can be extremely valuable during complex visual analytics tasks. We present an exploratory study of a system designed to support collaborative visual analysis tasks on a digital tabletop display. Fifteen participant pairs employed Cambiera, a visual analytics system, to solve a problem involving 240 digital documents. Our analysis, supported by observations, system logs, questionnaires, and interview data, explores how pairs approached the problem around the table. We contribute a unique, rich understanding of how users worked together around the table and identify eight types of collaboration styles that can be used to identify how closely people work together while problem solving. We show how the closeness of teams’ collaboration and communication influenced how they performed on the task overall. We further discuss the role of the tabletop for visual analytics tasks and derive design implications for future co-located collaborative tabletop problem solving systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/TVCG.2011.287DOI Listing
May 2012

Glycolysis in the african trypanosome: targeting enzymes and their subcellular compartments for therapeutic development.

Mol Biol Int 2011 11;2011:123702. Epub 2011 Apr 11.

Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.

Subspecies of the African trypanosome, Trypanosoma brucei, which cause human African trypanosomiasis, are transmitted by the tsetse fly, with transmission-essential lifecycle stages occurring in both the insect vector and human host. During infection of the human host, the parasite is limited to using glycolysis of host sugar for ATP production. This dependence on glucose breakdown presents a series of targets for potential therapeutic development, many of which have been explored and validated as therapeutic targets experimentally. These include enzymes directly involved in glucose metabolism (e.g., the trypanosome hexokinases), as well as cellular components required for development and maintenance of the essential subcellular compartments that house the major part of the pathway, the glycosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4061/2011/123702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195984PMC
November 2011

Glycerol 3-phosphate alters Trypanosoma brucei hexokinase activity in response to environmental change.

J Biol Chem 2011 Sep 3;286(38):33150-7. Epub 2011 Aug 3.

Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634, USA.

The African trypanosome, Trypanosoma brucei, compartmentalizes some metabolic enzymes within peroxisome-like organelles called glycosomes. The amounts, activities, and types of glycosomal enzymes are modulated coincident with developmental and environmental changes. Pexophagy (fusion of glycosomes with acidic lysosomes) has been proposed to facilitate this glycosome remodeling. Here, we report that, although glycosome-resident enzyme T. brucei hexokinase 1 (TbHK1) protein levels are maintained during pexophagy, acidification inactivates the activity. Glycerol 3-phosphate, which is produced in vivo by a glycosome-resident glycerol kinase, mitigated acid inactivation of lysate-derived TbHK activity. Using recombinant TbHK1, we found that glycerol 3-P influenced enzyme activity at pH 6.5 by preventing substrate and product inhibition by ATP and ADP, respectively. Additionally, TbHK1 inhibition by the flavonol quercetin (QCN) was partially reversed by glycerol 3-P at pH 7.4, whereas at pH 6.5, enzyme activity in the presence of QCN was completely maintained by glycerol 3-P. However, glycerol 3-P did not alter the interaction of QCN with TbHK1, as the lone Trp residue (Trp-177) was quenched under all conditions tested. These findings suggest potential novel mechanisms for the regulation of TbHK1, particularly given the acidification of glycosomes that can be induced under a variety of parasite growth conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.235705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190947PMC
September 2011

Quercetin, a fluorescent bioflavanoid, inhibits Trypanosoma brucei hexokinase 1.

Exp Parasitol 2011 Feb 11;127(2):423-8. Epub 2010 Nov 11.

Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.

Hexokinases from the African trypanosome, Trypanosoma brucei, are attractive targets for the development of anti-parasitic drugs, in part because the parasite utilizes glycolysis exclusively for ATP production during the mammalian infection. Here, we have demonstrated that the bioflavanoid quercetin (QCN), a known trypanocide, is a mixed inhibitor of Trypanosoma brucei hexokinase 1 (TbHK1) (IC(50) = 4.1 ± 0.8μM). Spectroscopic analysis of QCN binding to TbHK1, taking advantage of the intrinsically fluorescent single tryptophan (Trp177) in TbHK1, revealed that QCN quenches emission of Trp177, which is located near the hinge region of the enzyme. ATP similarly quenched Trp177 emission, while glucose had no impact on fluorescence. Supporting the possibility that QCN toxicity is a consequence of inhibition of the essential hexokinase, in live parasites QCN fluorescence localizes to glycosomes, the subcellular home of TbHK1. Additionally, RNAi-mediated silencing of TbHK1 expression expedited QCN induced death, while over-expressing TbHK1 protected trypanosomes from the compound. In summary, these observations support the suggestion that QCN toxicity is in part attributable to inhibition of the essential TbHK1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exppara.2010.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025057PMC
February 2011

A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity.

PLoS Negl Trop Dis 2010 Apr 13;4(4):e659. Epub 2010 Apr 13.

University of Pittsburgh Drug Discovery Institute and Pittsburgh Molecular Libraries Screening Center, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

Background: The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the gamma-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.

Methodology/principal Findings: Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were approximately 20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03
Conclusions/significance: The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0000659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854128PMC
April 2010

Trypanosoma brucei AMP-activated kinase subunit homologs influence surface molecule expression.

Exp Parasitol 2009 Nov 6;123(3):250-7. Epub 2009 Aug 6.

Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.

The African trypanosome, Trypanosoma brucei, can gauge its environment by sensing nutrient availability. For example, procyclic form (PF) trypanosomes monitor changes in glucose levels to regulate surface molecule expression, which is important for survival in the tsetse fly vector. The molecular connection between glycolysis and surface molecule expression is unknown. Here we partially characterize T. brucei homologs of the beta and gamma subunits of the AMP-activated protein kinase (AMPK), and determine their roles in regulating surface molecule expression. Using flow cytometry and mass spectrometry, we found that TbAMPKbeta or TbAMPKgamma-deficient parasites express both of the major surface molecules, EP- and GPEET-procyclin, with the latter being a form that is expressed when glucose is low such as in the tsetse fly. Last, we have found that the putative scaffold component of the complex, TbAMPKbeta, fractionates with organellar components and colocalizes in part with a glycosomal marker as well as the flagellum of PF parasites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exppara.2009.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774744PMC
November 2009

Assembly of heterohexameric trypanosome hexokinases reveals that hexokinase 2 is a regulable enzyme.

J Biol Chem 2008 May 3;283(22):14963-70. Epub 2008 Apr 3.

Department of Genetics and Biochemistry, Clemson University, 51 New Cherry Street, Clemson, SC 29634, USA.

Glycolysis is essential to Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness in humans and nagana in cattle. Hexokinase (HK), the first enzyme in glycolysis, catalyzes the phosphorylation of glucose to form glucose 6-phosphate. T. brucei harbors two HKs that are 98% identical at the amino acid level, T. brucei hexokinase 1 (TbHK1) and TbHK2. Recombinant TbHK1 (rTbHK1) has HK activity, whereas rTbHK2 does not. Unlike other eukaryotic HKs, TbHK1 is not subject to inhibition by ADP and glucose 6-phosphate. However, TbHK1 is inhibited by myristate, a critical fatty acid in T. brucei biology. We report here that rTbHKs, similar to authentic TbHK, form oligomers. Myristate dissociated these assemblies when incubated with either ATP or glucose. Furthermore, oligomer disruption was reversible by removal of myristate. Mixing of rTbHK1 and rTbHK2 monomers followed by reassembly yielded enzyme with an approximately 3-fold increase in specific activity compared with similarly treated rTbHK1 alone. Surprisingly, reassembly of rTbHK2 with an inactive rTbHK1 variant yielded an active HK, revealing for the first time that rTbHK2 is competent for HK activity. Finally, pyrophosphate inhibits active reassembled rTbHK2 oligomers but not oligomeric rTbHK1, suggesting that the two enzymes have distinct regulatory mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M802124200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258875PMC
May 2008

The anti-trypanosomal agent lonidamine inhibits Trypanosoma brucei hexokinase 1.

Mol Biochem Parasitol 2008 Apr 3;158(2):202-7. Epub 2008 Jan 3.

Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, United States.

Glycolysis is essential to the parasitic protozoan Trypanosoma brucei. The first step in this metabolic pathway is mediated by hexokinase, an enzyme that transfers the gamma-phosphate of ATP to a hexose. The T. brucei genome (TREU927/4 GUTat10.1) encodes two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. Our previous efforts have revealed that TbHK2 is an important regulator of TbHK1 in procyclic form parasites. Here, we have found through RNAi that TbHK1 is essential to the bloodstream form parasite. Silencing the gene for 4 days reduces cellular hexokinase approximately 60% and leads to parasite death. Additionally, we have found that the recombinant enzyme is inhibited by lonidamine (IC(50)=850 microM), an anti-cancer drug that targets tumor hexokinases. This agent also inhibits HK activity from whole parasite lysate (IC(50)=965 microM). Last, lonidamine is toxic to cultured bloodstream form parasites (LD(50)=50 microM) and procyclic form parasites (LD(50)=180 microM). Interestingly, overexpression of TbHK1 protects PF parasites from lonidamine. These studies provide genetic evidence that TbHK1 is a valid therapeutic target while identifying a potential molecular target of the anti-trypanosomal agent lonidamine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molbiopara.2007.12.013DOI Listing
April 2008

Residues in an ATP binding domain influence sugar binding in a trypanosome hexokinase.

Biochem Biophys Res Commun 2008 Jan 9;365(3):420-5. Epub 2007 Nov 9.

Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.

Trypanosoma brucei harbors two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. We previously found that recombinant TbHK1 (rTbHK1) has hexokinase activity, while rTbHK2 has not, a finding attributed to differences in the C-termini of the proteins. Sequence analysis suggests that the C-termini of TbHKs are part of a newly identified conserved motif found in other eukaryotic hexokinases. Here, we have explored the role of tail residues in the differences in catalytic activity between TbHK1 and TbHK2. Our studies reveal that tail residues D454, F462, M466, and N469 are essential for HK activity while both I458 and V468 are required for catalysis and substrate specificity. To activate rTbHK2, all of the residues important for activity in rTbHK1 (D454, V458, F462, M466, V468, and N469) were required. These results indicate that the overall structure of the C-terminal tail influences the HK activity of rTbHK1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2007.10.192DOI Listing
January 2008

Activity of a second Trypanosoma brucei hexokinase is controlled by an 18-amino-acid C-terminal tail.

Eukaryot Cell 2006 Dec 6;5(12):2014-23. Epub 2006 Oct 6.

Department of Genetics and Biochemistry, Clemson University, 214 Biosystems Research Complex, 51 New Cherry Street, Clemson, SC 29634, USA.

Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2-/- procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 +/- 0.02 mM and 0.28 +/- 0.1 mM, respectively. The k(cat) value for TbHK1 is 2.9 x 10(4) min(-1). TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 microM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/EC.00146-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694814PMC
December 2006

Neospora caninum expresses an unusual single-domain Kazal protease inhibitor that is discharged into the parasitophorous vacuole.

Int J Parasitol 2004 May;34(6):693-701

W. Harry Feinstein Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.

Serine protease inhibitors have been implicated in viral and parasite pathogenesis through their ability to inhibit apoptosis, provide protection against digestive enzymes in the gut and dictate host range specificity. Two Kazal family serine protease inhibitors from the obligate intracellular parasite Toxoplasma gondii (TgPI-1 and TgPI-2) have been characterised previously. Here, we describe the identification and initial characterisation of a novel Kazal inhibitor, NcPI-S, from a closely related apicomplexan parasite, Neospora caninum. Unlike the multidomain inhibitors identified in T. gondii, NcPI-S is a single domain inhibitor bearing a methionine in the position (P1) that typically dictates specificity for target proteases. Based on this, NcPI-S was predicted to inhibit elastase, chymotrypsin and subtilisin. However, we found that recombinant NcPI-S inhibited subtilisin very well, with little or no activity against elastase or chymotrypsin. NcPI-S localises to the dense granules and is secreted into the parasitophorous vacuole. Finally, antibodies raised against recombinant NcPI-S recognise two polypeptides in an N. caninum lysate, one with a molecular mass approximately 11 kDa and another at approximately 20 kDa. This, along with mass spectrometry analysis of recombinant NcPI-S, suggests that the inhibitor is expressed as a dimer in the parasite.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpara.2004.01.004DOI Listing
May 2004

Identification and partial characterization of a second Kazal inhibitor in Toxoplasma gondii.

Mol Biochem Parasitol 2003 Apr;128(1):119-22

The W Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N Wolfe Street, Baltimore, MD 21205, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0166-6851(03)00051-3DOI Listing
April 2003
-->