Education and Career Development

This presentation describes educational programs for trainees and faculty that are offered through the UMCCTS

Reconciling contradictory findings: Glucose transporter 1 (GLUT1) functions as an oligomer of allosteric, alternating access transporters

Recent structural studies suggest that glucose transporter 1 (GLUT1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple beta-D-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single beta-D-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the Km(app) for 2-deoxy-D-glucose uptake, eliminated cis-allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not trans-allostery (uptake stimulation by subsaturating cytochalasin B). Cis-allostery persisted, but trans-allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated cis- allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) cis-allostery is mediated by intra-subunit interactions and 2) trans-allostery requires inter-subunit interactions

Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of GLUT1-mediated sugar uptake

The anti-oxidant, flavonoid-rich content of red wine and green tea is reported to offer protection against cancer, cardiovascular disease and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport raising the possibility that their interaction with GLUT1 and subsequent, downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure/function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake while higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUTs 1, 3 and 4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that ECG is more selective for GLUT1 while EGCG is less isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding whereas EGC shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer\u27s

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 mum but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 mum but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 - GLUT1 - neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites

Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double strand breaks (DSB) which co-localized with 'replication factories' and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro. These data identify RS as a cancer stem cell-specific target with significant clinical potential

The place of agricultural research in the development of sub?Saharan Africa

SUMMARY The agricultural research policy issue in sub?Saharan Africa can be addressed by seeking to explain a paradox. Rates of return to agricultural research have been shown to be large. By the standards of the developing world, expenditure on such research is high in sub?Saharan Africa, yet agricultural growth has been notoriously slow. There are two explanations. First, there is less agricultural research in most of sub?Saharan Africa than there appears to be. Second, it is less good at producing output?orientated results than other poor countries. Much of the problem lies in its nature. Difficulties in attaining critical mass are aggravated by higher turnover, loss of benefits to neighbouring countries and dispersion of scientists. The latter raises questions of research relevance and congruence. SOMMAIRE On peut traiter du problème de la politique de recherche agricole en Afrique subsaharienne en essayant d'expliquer un paradoxe. On a montré que le taux de rentabilité de la recherche agricole est important. Comparé au niveau des pays en développement, les dépenses pour de telles recherches en Afrique subsaharienne sont élevées, pourtant la croissance agricole a été notoirement lente. Il y a deux explications: d'abord, il y a moins de recherche agricole dans la plupart de l'Afrique subsaharienne qu'il n'y paraît. Deuxièmement, ces recherches ne sont pas aussi utiles, quand il s'agit de résultats orientés vers le rendement, que celles d'autres pays pauvres. La plus grande partie du problème vient de leur nature. Les difficultés pour atteindre la masse critique sont aggravées par un important roulement, des pertes de bénéfices allant aux pays voisins et la dispersion des savants. Ce dernier point pose des questions de pertinence et de convenance de la recherche. RESUMEN El tema de la política de investigación agrícola en la región del sub?Sahara del Africa puede ser encarado mediante un intento por explicar una paradoja. Las tasas de retorno de la investigación agricola han demostrado ser altas. De acuerdo a los patrones del mundo desarrollado el gasto en estas investigaciones es alto en la región del sub?Sahara del Africa y sin embargo el crecimiento agrícola ha sido notoriamente lento. Al respecto se ha dado dos explicaciones. En primer lugar hay menos investigación agrícola en gran parte de la región del sub?Sahara del Africa de lo que aparece. En segundo lugar, en comparación con otros países pobres tienen una orientación menos marcada hacia la producción de resultados. Parte importante del problema descansa en su naturaleza. Las dificultades para conseguir una acumulación de resultados se ven agravadas por una alta rotación de personal, por la pérdida de beneficios hacia los paises vecinos y por la dispersión de científicos. Esto último plantea interrogantes acerca de la relevancia y la congruencia de la investigación

Pharmacokinetics, safety and tolerability of olaparib and temozolomide for recurrent glioblastoma: results of the phase I OPARATIC trial

Background: The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide chemotherapy in pre-clinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side-effects of temozolomide. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib, and assess the safety and tolerability of its combination with temozolomide. Methods: Pre-clinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose temozolomide in a 3+3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumour core and tumour margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. Results: Olaparib was a substrate for multi-drug resistance protein-1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients, median 496nM) and 21/21 tumor margin specimens (9 patients, median 512.3nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150mg (3 days/week) with TMZ 75mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression-free at 6 months. Olaparib radiosensitized six glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. Conclusions: Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better pre-clinical models

Energy Solutions, Neo-Liberalism, and Social Diversity in Toronto, Canada

In response to the dominance of green capitalist discourses in Canada’s environmental movement, in this paper, we argue that strategies to improve energy policy must also provide mechanisms to address social conflicts and social disparities. Environmental justice is proposed as an alternative to mainstream environmentalism, one that seeks to address systemic social and spatial exclusion encountered by many racialized immigrants in Toronto as a result of neo-liberal and green capitalist municipal policy and that seeks to position marginalized communities as valued contributors to energy solutions. We examine Toronto-based municipal state initiatives aimed at reducing energy use while concurrently stimulating growth (specifically, green economy/green jobs and ‘smart growth’). By treating these as instruments of green capitalism, we illustrate the utility of environmental justice applied to energy-related problems and as a means to analyze stakeholders’ positions in the context of neo-liberalism and green capitalism, and as opening possibilities for resistance