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[Display omitted] Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features but apparently differ in severity. The two known carriers of the Met374Arg variant had early childhood disease onset and more severe motor, cognitive, and neuropsychiatric deficits than any known carriers of the Ile212Phe variant, whose symptoms were first apparent in adolescence. Here, we evaluated if differences in the function of the two variants in cultured cells could explain differing pathogenicity. Both variants were expressed less abundantly than the wild type receptor and exhibited loss of agonist-induced arrestin binding, but differences in expression and arrestin binding between the variants were minor. Basal and agonist-induced activation of heterotrimeric G i/o/z proteins, however, showed clear differences; agonists were generally more potent at Met374Arg than at the Ile212Phe or wild type variants. Furthermore, all Gα subtypes tested were constitutively activated more by Met374Arg than by Ile212Phe. Met374Arg produced greater constitutive inhibition of cyclic AMP accumulation than Ile212Phe or the wild type D2 receptor. Met374Arg and Ile212Phe were more sensitive to thermal inactivation than the wild type D2 receptor, as reported for other constitutively active receptors, but Ile212Phe was affected more than Met374Arg. Additional pharmacological characterization suggested that the mutations differentially affect the shape of the agonist binding pocket and the potency of dopamine, norepinephrine, and tyramine. Molecular dynamics simulations provided a structural rationale for enhanced constitutive activation and agonist potency. Enhanced constitutive and agonist-induced G protein-mediated signaling likely contributes to the pathogenicity of these novel variants. [ABSTRACT FROM AUTHOR]

Background: We describe a 4‐generation Dutch pedigree with a unique dominantly inherited clinical phenotype of a combined progressive chorea and cervical dystonia carrying a novel heterozygous dopamine D2 receptor (DRD2) variant. Objectives: The objective of this study was to identify the genetic cause of the disease and to further investigate the functional consequences of the genetic variant. Methods: After detailed clinical and neurological examination, whole‐exome sequencing was performed. Because a novel variant in the DRD2 gene was found as the likely causative gene defect in our pedigree, we sequenced the DRD2 gene in a cohort of 121 Huntington‐like cases with unknown genetic cause (Germany). Moreover, functional characterization of the DRD2 variant included arrestin recruitment, G protein activation, and G protein‐mediated inhibition of adenylyl cyclase determined in a cell model, and G protein‐regulated inward‐rectifying potassium channels measured in midbrain slices of mice. Result: We identified a novel heterozygous variant c.634A > T, p.Ile212Phe in exon 5 of DRD2 that cosegregated with the clinical phenotype. Screening of the German cohort did not reveal additional putative disease‐causing variants. We demonstrated that the D2S/L‐I212F receptor exhibited increased agonist potency and constitutive activation of G proteins in human embryonic kidney 239 cells as well as significantly reduced arrestin3 recruitment. We further showed that the D2S‐I212F receptor exhibited aberrant receptor function in mouse midbrain slices. Conclusions: Our results support an association between the novel p.Ile212Phe variant in DRD2, its modified D2 receptor activity, and the hyperkinetic movement disorder reported in the 4‐generation pedigree. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

In Leishmania mexicana parasites, a unique glucose transporter, LmxGT1, is selectively targeted to the flagellar membrane, suggesting a possible sensory role that is often associated with ciliary membrane proteins. Expression of LmxGT1 is down-regulated ~20-fold by increasing cell density but is up-regulated ~50-fold by depleting glucose from the medium, and the permease is strongly down-regulated when flagellated insect-stage promastigotes invade mammalian macrophages and transform into intracellular amastigotes. Regulation of LmxGT1 expression by glucose and during the lifecycle operates at the level of protein stability. Significantly, a Δlmxgt1 null mutant, grown in abundant glucose, undergoes catastrophic loss of viability when parasites deplete glucose from the medium, a property not exhibited by wild-type or add-back lines. These results suggest that LmxGT1 may function as a glucose sensor that allows parasites to enter the stationary phase when they deplete glucose and that in the absence of this sensor, parasites do not maintain viability when they run out of glucose. However, alternate roles for LmxGT1 in monitoring glucose availability are considered. The absence of known sensory receptors with defined ligands and biologic functions in Leishmania and related kinetoplastid parasites underscores the potential significance of these observations. [ABSTRACT FROM AUTHOR]

Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that L-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. [ABSTRACT FROM AUTHOR]

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle. [ABSTRACT FROM AUTHOR]

The genome of Leishmania mexicana encompasses a cluster of three glucose transporter genes designated LmxGT1, LmxGT2 and LmxGT3. Functional and genetic studies of a cluster null mutant (Δ lmxgt1-3) have dissected the roles of these proteins in Leishmania metabolism and virulence. However, null mutants were recovered at very low frequency, and comparative genome hybridizations revealed that Δ lmxgt1-3 mutants contained a linear extrachromosomal 40 kb amplification of a region on chromosome 29 not amplified in wild type parasites. These data suggested a model where this 29-40k amplicon encoded a second site suppressor contributing to parasite survival in the absence of GT1-3 function. To test this, we quantified the frequency of recovery of knockouts in the presence of individual overexpressed open reading frames covering the 29-40k amplicon. The data mapped the suppressor activity to PIFTC3, encoding a component of the intraflagellar transport pathway. We discuss possible models by which PIFTC3 might act to facilitate loss of GTs specifically. Surprisingly, by plasmid segregation we showed that continued PIFTC3 overexpression was not required for Δ lmxgt1-3 viability. These studies provide the first evidence that genetic suppression can occur by providing critical biological functions transiently. This novel form of genetic suppression may extend to other genes, pathways and organisms. [ABSTRACT FROM AUTHOR]

Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96- M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3). of which TgST2 is expressed at its surface, whereas T9ST1 and T9ST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Δgst2, the modest impairment in Δggt1 and Δggt1/Δgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Δggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1 -complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Δggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Δggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells. [ABSTRACT FROM AUTHOR]

A glucose transporter null mutant of the parasitic protozoan Leishmania mexicana, in which three linked glucose transporter genes have been deleted by targeted gene replacement, is unable to replicate as amastigote forms within phagolysomes of mammalian host macrophages and is avirulent. Spontaneous suppressors of the null mutant have been isolated that partially restore replication of parasites within macrophages. These suppressor mutants have amplified the gene for an alternative hexose transporter, the LmGT4 permease (previously called the D2 permease), on a circular extrachromosomal element, and they overexpress LmGT4 mRNA and protein. The suppressors have also regained the ability to transport hexoses, and they have reverted other phenotypes of the null mutant exhibiting enhanced resistance to oxidative killing, heat shock and starvation for nutrients, as well as augmented levels of the storage carbohydrate β-mannan, increased cell size and increased growth as insect stage promastigotes compared with the unsuppressed mutant. Complementation of the null mutant with the LmGT4 gene on a multicopy episomal expression vector also reverted these phenotypes, confirming that suppression results from amplification of the LmGT4 gene. These results underscore the importance of hexose transporters for the infectious stage of the parasite life cycle. [ABSTRACT FROM AUTHOR]

Abstract: Glucose is a major source of energy and carbon in promastigotes of Leishmania mexicana, and its uptake is mediated by three glucose transporters whose genes are encoded within a single cluster. A null mutant in which the glucose transporter gene cluster was deleted by homologous gene replacement was generated previously and shown to grow more slowly than wild type promastigotes but not to be viable as amastigotes in primary tissue culture macrophages or in axenic culture. Further phenotypic characterization demonstrates that the null mutant is unable to import glucose, mannose, fructose, or galactose and that each of the three glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, is capable of transporting each of these hexoses. Complementation of the null mutant with each isoform is able to restore growth in each of the four hexoses to wild type levels. Null mutant promastigotes are reduced in size to about 2/3 the volume of wild type parasites. In addition, the null mutants are significantly more sensitive to oxidative stress than their wild type counterparts. These results underscore the importance of glucose transporters in the parasite life cycle and suggest reasons for their non-viability in the disease-causing amastigote stage. [Copyright &y& Elsevier]

Leishmania mexicana are parasitic protozoa that express a variety of glycoconjugates that play important roles in their biology as well as the storage carbohydrate β-mannan, which is an essential virulence factor for survival of intracellular amastigote forms in the mammalian host. Glucose transporter null mutants, which are viable as insect form promastigotes but not as amastigotes, do not take up glucose and other hexoses but are still able to synthesize these glycoconjugates and β-mannan, although at reduced levels. Synthesis of these carbohydrate-containing macromolecules could be accounted for by incorporation of non-carbohydrate precursors into carbohydrates by gluconeogenesis. However, the significantly reduced level of the virulence factor β-mannan in the glucose transporter null mutants compared with wild-type parasites may contribute to the non-viability of these null mutants in the disease-causing amastigote stage of the life cycle. [ABSTRACT FROM AUTHOR]

Studies the genetic characterization of glucose transporter function in Leishmania mexicana. Transport properties of DeltaImgt knockout promastigotes; Growth properties of DeltaImgt promastigotes.

R4511 --> A novel DRD2 variant with a mutation in exon 5 of DRD2(c.634A>T; p.I212F)has been identified in a Dutch family. Ile212 is at the N‐terminus of the D2 receptor (D2R) 3rd cytoplasmic loop. D2‐I212F is associated with a novel hyperkinetic movement disorder characterized by progressive chorea and cervical dystonia. We hypothesized that determining the effect of the mutation on D2R function might explain how D2‐I212F causes the movement disorder. Molecular dynamics (MD) simulations of human D2R models suggested that the presence of Phe212 in D2R separates the side chains of Arg132, at the cytoplasmic end of the 3rd transmembrane domain (TM3), and Glu368, at the cytoplasmic end of TM6, spontaneously breaking the ionic lock in the inactive D2‐I212F. This ionic lock contributes to maintaining unliganded receptors in an inactive conformation, and it is broken in agonist‐activated receptors. Conversely, preventing formation of the lock frequently creates a constitutively active receptor. In vitro and ex vivo studies supported the hypothesis that D2‐I212F is a constitutively active receptor: We expressed D2L/S‐I212F in HEK293 cells and assayed activation of Gαi1 and GαoA by bioluminescence resonance energy transfer. We observed increased quinpirole potency to activate Gαi1 by D2L/S‐I212F (2.8 or 3.5 nM for D2L or D2S, respectively) compared to D2L/S‐WT (19 or 27 nM) whereas quinpirole potency for activating GαoA was not substantially changed by the I212F mutation. On the other hand, D2L/S‐I212F exhibited increased basal activation of both Gαi/o proteins compared to D2L/S‐WT (set as 0%); basal GαoA activation by D2L/S‐I212F (43 or 57% of maximal stimulation, respectively) was significantly higher than D2L/S‐I212F‐mediated basal Gαi1 activation (25% and 35%, respectively). We then used AAV to express Cre recombinase‐regulated D2S‐WT or D2S‐I212F in dopamine neurons of D2 autoreceptor knockout mice and characterized D2R activation of G protein‐regulated inward‐rectifying potassium channels (GIRKs). When CyHQ‐sulpiride (5 μM) was circulated over a midbrain slice, photolytic release of sulpiride produced a small inhibition of a tonic GIRK current in cells expressing D2‐WT (‐9 pA), and a much larger inhibition in cells expressing D2‐I212F (‐62 pA). The enhanced inhibition of tonic current by the mutant receptor could reflect constitutive activation of G proteins or a heightened sensitivity of the mutant to dopamine. To distinguish between these possibilities, midbrain slices were treated with reserpine to deplete endogenous dopamine. This treatment abolished the response to sulpiride in cells expressing D2‐WT and greatly decreased the response in cells expressing D2‐I212F, indicating that most of the tonic current was due to endogenous dopamine to which D2‐I212F may be more sensitive, but that D2‐I212F also displayed some constitutive activity in the presumed absence of dopamine. Thus, our in silico, in vitro, and ex vivo studies all indicate that the mutation enhances constitutive and agonist‐induced activation of G proteins by D2R [ABSTRACT FROM AUTHOR]

Glucose is considered essential for erythrocytic stages of the malaria parasite, Plasmodium falciparum. Importance of sugar and its permease for hepatic and sexual stages of Plasmodium, however, remains elusive. Moreover, increasing global resistance to current antimalarials necessitates the search for novel drugs. Here, we reveal that hexose transporter 1 (HT1) of Plasmodium berghei can transport glucose (Km~87 μM), mannose (Ki~93 μM), fructose (Ki~0.54 mM), and galactose (Ki~5 mM) in Leishmania mexicana mutant and Xenopus laevis; and, therefore, is functionally equivalent to HT1 of P. falciparum (Glc, Km~175 μM; Man, Ki~ μ276M; Fru, Ki~1.25 mM; Gal, Ki5.86mM). Notably, a glucose analog, C3361, attenuated hepatic (IC50~15 μM) and ookinete development of P. berghei. The PbHT1 could be ablated during intraerythrocytic stages only by concurrent complementation with PbHT1-HA or PfHT1. Together; these results signify that PbHT1 and glucose are required for the entire life cycle of P. berghei. Accordingly, PbHT1 is expressed in the plasma membrane during all parasite stages. To permit a high-throughput screening of PfHT1 inhibitors and their subsequent in vivo assessment, we have generated Saccharomyces cerevisiae mutant expressing codon-optimized PfHT1, and a PfHT1-dependent Δpbht1 parasite strain. This work provides a platform to facilitate the development of drugs against malaria, and it suggests a disease-control aspect by reducing parasite transmission. [ABSTRACT FROM AUTHOR]

Abstract: Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of Leishmania mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose. [ABSTRACT FROM AUTHOR]