Lothrop AP et al. (2009) Biochemistry "No selenium required: reactions catalyzed by mammalian thioredoxin reductase that are independent of a selenocysteine residue."

Hondal RJ, Ruggles EL, Lothrop AP

[Biochemistry, 2009]

Mammalian thioredoxin reductase (TR) contains a rare selenocysteine (Sec) residue in a conserved redox-active tetrapeptide of sequence Gly-Cys(1)-Sec(2)-Gly. The high chemical reactivity of the Sec residue is thought to confer broad substrate specificity to the enzyme. In addition to utilizing thioredoxin (Trx) as a substrate, other substrates are protein disulfide isomerase, glutaredoxin, glutathione peroxidase, NK-lysin/granulysin, HIV Tat protein, H(2)O(2), lipid hydroperoxides, vitamin K, ubiquinone, juglone, ninhydrin, alloxan, dehydroascorbate, DTNB, lipoic acid/lipoamide, S-nitrosoglutathione, selenodiglutathione, selenite, methylseleninate, and selenocystine. Here we show that the Cys(2) mutant enzyme or the N-terminal reaction center alone can reduce Se-containing substrates selenocystine and selenite with only slightly less activity than the wild-type enzyme, in stark contrast to when Trx is used as the substrate when the enzyme suffers a 175-550-fold reduction in k(cat). Our data support the use of alternative mechanistic pathways for the Se-containing substrates that bypass a critical ring-forming step when Trx is the substrate. We also show that lipoic acid can be reduced through a Sec-independent mechanism that involves the N-terminal reaction center. These results show that the broad substrate specificity of the mammalian enzyme is not due to the presence of the rare Sec residue but is due to the catalytic power of the N-terminal reaction center. We hypothesize that the N-terminal reaction center can reduce substrates (i) with good leaving groups such as DTNB, (ii) that are highly electrophilic such as selenite, or (iii) that are activated by strain such as lipoic acid/lipoamide. We also show that the absence of Sec only changed the IC(50) for aurothioglucose by a factor of 1.7 in the full-length mammalian enzyme (83-142 nM), but surprisingly the truncated enzyme showed much stronger inhibition (25 nM). This contrasts with auranofin, where the absence of Sec more strongly perturbed inhibition.

Lothrop AP et al. (2014) Biochemistry "Why is mammalian thioredoxin reductase 1 so dependent upon the use of selenium?"

Lothrop AP, Snider GW, Ruggles EL, Hondal RJ

[Biochemistry, 2014]

Cytosolic thioredoxin reductase 1 (TR1) is the best characterized of the class of high-molecular weight (Mr) thioredoxin reductases (TRs). TR1 is highly dependent upon the rare amino acid selenocysteine (Sec) for the reduction of thioredoxin (Trx) and a host of small molecule substrates, as mutation of Sec to cysteine (Cys) results in a large decrease in catalytic activity for all substrate types. Previous work in our lab and others has shown that the mitochondrial TR (TR3) is much less dependent upon the use of Sec for the reduction of small molecules. The Sec-dependent substrate utilization behavior of TR1 may be the exception and not the rule as we show that a variety of high-Mr TRs from other organisms, including Drosophila melanogaster, Caenorhabditis elegans, and Plasmodium falciparum, do not require Sec to reduce small molecule substrates, including 5,5'-dithiobis(2-nitrobenzoic acid), lipoic acid, selenite, and selenocystine. The data show that high-Mr TRs can be divided into two groups based upon substrate utilization patterns: a TR1 group and a TR3-like group. We have constructed mutants of TR3-like enzymes from mouse, D. melanogaster, C. elegans, and P. falciparum, and the kinetic data from these mutants show that these enzymes are less dependent upon the use of Sec for the reduction of substrates. We posit that the mechanistic differences between TR1 and the TR3-like enzymes in this study are due to the presence of a "guiding bar", amino acids 407-422, found in TR1, but not TR3-like enzymes. The guiding bar, proposed by Becker and co-workers [Fritz-Wolf, K., Urig, S., and Becker, K. (2007) The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis. J. Mol. Biol. 370, 116-127], restricts the motion of the C-terminal tail containing the C-terminal Gly-Cys-Sec-Gly, redox active tetrapeptide so that only this C-terminal redox center can be reduced by the N-terminal redox center, with the exclusion of most other substrates. This makes TR1 highly dependent upon the use of Sec because the selenium atom is responsible for both accepting electrons from the N-terminal redox center and donating them to the substrate in this model. Loss of both Se-electrophilicity and Se-nucleophilicity in the Sec Cys mutant of TR1 greatly reduces catalytic activity. TR3-like enzymes, in contrast, are less dependent upon the use of Sec because the absence of the guiding bar in these enzymes allows for greater access of the substrate to the N-terminal redox center and because they can make use of alternative mechanistic pathways that are not available to TR1.

Boehm M et al. (2001) Mol Biol Cell "Adaptins: the final recount."

Bonifacino JS, Boehm M

[Mol Biol Cell, 2001]

Adaptins are subunits of adaptor protein (AP) complexes involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles. In this article, we report the results of a survey for adaptins from sequenced genomes including those of man, mouse, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the plant Arabidopsis thaliana, and the yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We find that humans, mice, and Arabidopsis thaliana have four AP complexes (AP-1, AP-2, AP-3, and AP-4), whereas D. melanogaster, C. elegans, S. cerevisiae, and S. pombe have only three (AP-1, AP-2, and AP-3). Additional diversification of AP complexes arises from the existence of adaptin isoforms encoded by distinct genes or resulting from alternative splicing of mRNAs. We complete the assignment of adaptins to AP complexes and provide information on the chromosomal localization, exon-intron structure, and pseudogenes for the different adaptins. In addition, we discuss the structural and evolutionary relationships of the adaptins and the genetic analyses of their function. Finally, we extend our survey to adaptin-related proteins such as the GGAs and stonins, which contain domains homologous to the adaptins.

Li P et al. (2016) Neuron "Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity."

Li P, Jorgensen EM, Shen K, Merrill SA

[Neuron, 2016]

The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C.elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools.

Peden AA et al. (2004) J Cell Biol "Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins."

Oorschot V, Scheller RH, Klumperman J, Austin CD, Peden AA, Hesser BA

[J Cell Biol, 2004]

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3- and AP-1-bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3-positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3-deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.

Shafaq-Zadah M et al. (2012) Development "AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine."

Michaux G, Brocard L, Shafaq-Zadah M, Solari F

[Development, 2012]

Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium.

Perez-Gomez A et al. (2018) Nat Commun "A phenotypic Caenorhabditis elegans screen identifies a selective suppressor of antipsychotic-induced hyperphagia."

Osborn O, Petrascheck M, Perez-Gomez A, Carretero M, Solis G, Weber N, Titova V, Peterka V, To A

[Nat Commun, 2018]

Antipsychotic (AP) drugs are used to treat psychiatric disorders but are associated with significant weight gain and metabolic disease. Increased food intake (hyperphagia) appears to be a driving force by which APs induce weight gain but the mechanisms are poorly understood. Here we report that administration of APs to C. elegans induces hyperphagia by a mechanism that is genetically distinct from basal food intake. We exploit this finding to screen for adjuvant drugs that suppress AP-induced hyperphagia in C. elegans and mice. In mice AP-induced hyperphagia is associated with a unique hypothalamic gene expression signature that is abrogated by adjuvant drug treatment. Genetic analysis of this signature using C. elegans identifies two transcription factors, nhr-25/Nr5a2 and nfyb-1/NFYB to be required for AP-induced hyperphagia. Our study reveals that AP-induced hyperphagia can be selectively suppressed without affecting basal food intake allowing for novel drug discovery strategies to combat AP-induced metabolic side effects.

Bailey S et al. (2002) Structure "The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms."

Baker PJ, Bailey S, Abdelghany HM, Sedelnikova SE, Blackburn GM, Rafferty JB, McLennan AG

[Structure, 2002]

The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 Angstrom. and 1.8 Angstrom, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.

Bailey S et al. (2002) Acta Crystallogr D Biol Crystallogr "Crystallization of a complex of Caenorhabditis elegans diadenosine tetraphosphate hydrolase and a non-hydrolysable substrate analogue, AppCH2ppA."

Bailey S, Abdelghany HM, McLennan AG, Blackburn GM, Rafferty JB, Sedelnikova SE

[Acta Crystallogr D Biol Crystallogr, 2002]

The molecule diadenosine tetraphosphate (Ap(4)A) has been suggested to be a component of the cellular response to metabolic stress and/or, via the intracellular Ap(3)A/Ap(4)A ratio, to be involved in differentiation and apoptosis. Thus, the enzyme Ap(4)A hydrolase has a key metabolic role through regulation of the intracellular Ap(4)A levels. Crystals of this enzyme from the nematode Caenorhabditis elegans have been obtained in the presence of a non-hydrolysable substrate analogue, AppCH(2)ppA. The crystals belong to space group P2(1), unit-cell parameters a = 57.6, b = 36.8, c = 68.9 Angstrom, beta = 114.2degrees, and diffract to approximately 2.0 Angstrom. Determination of the structure of this complex will provide insights into the substrate specificity and catalytic activity of this class of enzymes.

Gillard G et al. (2015) Development "Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells."

Pecreaux J, Shafaq-Zadah M, Damaj R, Michaux G, Gillard G, Nicolle O

[Development, 2015]

E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis.