Congenital hypothyroidism mutations affect common folding and trafficking in the α/ß-hydrolase fold proteins.

The α/ß-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/ß-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/ß-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/ß-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/ß-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/ß-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination.

Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and ATF6 activation.

Although genetic variations in several genes encoding for synaptic adhesion proteins have been found to be associated with autism spectrum disorders, one of the most consistently replicated genes has been CNTNAP2, encoding for contactin-associated protein-like 2 (CASPR2), a multidomain transmembrane protein of the neurexin superfamily. Using immunofluorescence confocal microscopy and complementary biochemical techniques, we compared wild-type CASPR2 to 12 point mutations identified in individuals with autism. In contrast to the wild-type protein, localized to the cell surface, some of the mutants show altered cellular disposition. In particular, CASPR2-D1129H is largely retained in the endoplasmic reticulum (ER) in HEK-293 cells and in hippocampal neurons. BiP/Grp78, Calnexin and ERp57, key ER chaperones, appear to be responsible for retention of this mutant and activation of one signaling pathway of the unfolded protein response (UPR). The presence of this mutation also lowers expression and activates proteosomal degradation. A frame-shift mutation that causes a form of syndromic epilepsy (CASPR2-1253*), results in a secreted protein with seemingly normal folding and oligomerization. Taken together, these data indicate that CASPR2-D1129H has severe trafficking abnormalities and CASPR2-1253* is a secreted soluble protein, suggesting that the structural or signaling functions of the membrane tethered form are lost. Our data support a complex genetic architecture in which multiple distinct risk factors interact with others to shape autism risk and presentation.

Processing of cholinesterase-like α/ß-hydrolase fold proteins: alterations associated with congenital disorders.

The α/ß hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/ß hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/ß-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/ß-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/ß-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/ß-hydrolase fold.

Neuroligin trafficking deficiencies arising from mutations in the alpha/beta-hydrolase fold protein family.

Despite great functional diversity, characterization of the alpha/beta-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the alpha/beta-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.

Cis and trans actions of the cholinesterase-like domain within the thyroglobulin dimer.

Thyroglobulin (Tg, precursor for thyroid hormone synthesis) is a large secreted glycoprotein composed of upstream regions I-II-III, followed by the approximately 570 residue cholinesterase-like (ChEL) domain. ChEL has two identified functions: 1) homodimerization, and 2) binding to I-II-III that facilitates I-II-III oxidative maturation required for intracellular protein transport. Like its homologs in the acetylcholinesterase (AChE) family, ChEL possesses two carboxyl-terminal alpha-helices. We find that a Tg-AChE chimera (swapping AChE in place of ChEL) allows for dimerization with monomeric AChE, proving exposure of the carboxyl-terminal helices within the larger context of Tg. Further, we establish that perturbing trans-helical interaction blocks homodimerization of the Tg ChEL domain. Additionally, ChEL can associate with neuroligins (a related family of cholinesterase-like proteins), demonstrating potential for Tg cross-dimerization between non-identical partners. Indeed, when mutant rdw-Tg (Tg-G2298R, defective for protein secretion) is co-expressed with wild-type Tg, the two proteins cross-dimerize and secretion of rdw-Tg is partially restored. Moreover, we find that AChE and soluble neuroligins also can bind to the upstream Tg regions I-II-III; however, they cannot rescue secretion, because they cannot facilitate oxidative maturation of I-II-III. These data suggest that specific properties of distinct Tg ChEL mutants may result in distinct patterns of Tg monomer folding, cross-dimerization with wild-type Tg, and variable secretion behavior in heterozygous patients.

Folding anomalies of neuroligin3 caused by a mutation in the alpha/beta-hydrolase fold domain.

Proteins of the alpha/beta-hydrolase fold family share a common structural fold, but perform a diverse set of functions. We have been studying natural mutations occurring in association with congenital disorders in the alpha/beta-hydrolase fold domain of neuroligin (NLGN), butyrylcholinesterase (BChE), acetylcholinesterase (AChE). Starting from the autism-related R451C mutation in the alpha/beta-hydrolase fold domain of NLGN3, we had previously shown that the Arg to Cys substitution is responsible for endoplasmic reticulum (ER) retention of the mutant protein and that a similar trafficking defect is observed when the mutation is inserted at the homologous positions in AChE and BChE. Herein we show further characterization of the R451C mutation in NLGN3 when expressed in HEK-293, and by protease digestion sensitivity, we reveal that the phenotype results from protein misfolding. However, the presence of an extra Cys does not interfere with the formation of disulfide bonds as shown by reaction with PEG-maleimide and estimation of the molecular mass changes. These findings highlight the role of proper protein folding in protein processing and localization.

Extracellular zinc and zinc-citrate, acting through a putative zinc-sensing receptor, regulate growth and survival of prostate cancer cells.

Prostate Zn(2+) concentrations are among the highest in the body, and a marked decrease in the level of this ion is observed in prostate cancer. Extracellular Zn(2+) is known to regulate cell survival and proliferation in numerous tissues. In spite of this, a signaling role for extracellular Zn(2+) in prostate cancer has not been established. In the present study, we demonstrate that prostate metastatic cells are impermeable to Zn(2+), but extracellular Zn(2+) triggers a metabotropic Ca(2+) rise that is also apparent in the presence of citrate. Employing fluorescent imaging, we measured this activity in androgen-insensitive metastatic human cell lines, PC-3 and DU-145, and in mouse prostate tumor TRAMP-1 cells but not in androgen-sensitive LNCaP cells. The Ca(2+) response was inhibited by Galphaq and phospholipase C (PLC) inhibitors as well as by intracellular Ca(2+) store depletion, indicating that it is mediated by a Gq-coupled receptor that activates the inositol phosphate (IP(3)) pathway consistent with the previously identified zinc-sensing receptor (ZnR). Zn(2+)-dependent extracellular signal-regulated kinase and AKT activation, as well as enhanced Zn(2+)-dependent cell growth and survival, were observed in PC-3 cells that exhibit ZnR activity, but not in a ZnR activity-deficient PC-3 subline. Interestingly, application of Zn(2+)-citrate (Zn(2+)Cit), at physiological concentrations, was followed by a profound functional desensitization of extracellular Zn(2+)-dependent signaling and attenuation of Zn(2+)-dependent cell growth. Our results indicate that extracellular Zn(2+) and Zn(2+)Cit, by triggering or desensitizing ZnR activity, distinctly regulate prostate cancer cell growth. Thus, therapeutic strategies based either on Zn(2+) chelation or administration of Zn(2+)Cit may be effective in attenuating prostate tumor growth.

Carotenoids activate the antioxidant response element transcription system.

Epidemiologic studies have found an inverse association between consumption of tomato products and the risk of certain types of cancers. However, the mechanisms underlying this relationship are not completely understood. One mechanism that has been suggested is induction of phase II detoxification enzymes. Expression of phase II enzymes is regulated by the antioxidant response element (ARE) and the transcription factor Nrf2 (nuclear factor E2-related factor 2). In this study, we determined the role of this transcription system in the induction of phase II enzymes by carotenoids. We found that in transiently transfected cancer cells, lycopene transactivated the expression of reporter genes fused with ARE sequences. Other carotenoids such as phytoene, phytofluene, beta-carotene, and astaxanthin had a much smaller effect. An increase in protein as well as mRNA levels of the phase II enzymes NAD(P)H:quinone oxidoreductase and gamma-glutamylcysteine synthetase was observed in nontransfected cells after carotenoid treatment. Ethanolic extract of lycopene containing unidentified hydrophilic derivatives of the carotenoid activated ARE with similar potency to lycopene. The potency of the carotenoids in ARE activation did not correlate with their effect on intracellular reactive oxygen species and reduced glutathione level, which may indicate that ARE activation is not solely related to their antioxidant activity. Nrf2, which is found predominantly in the cytoplasm of control cells, translocated to the nucleus after carotenoid treatment. Interestingly, part of the translocated Nrf2 colocalized with the promyelocytic leukemia protein in the promyelocytic leukemia nuclear bodies. The increase in phase II enzymes was abolished by a dominant-negative Nrf2, suggesting that carotenoid induction of these proteins depends on a functional Nrf2 and the ARE transcription system.

Carotenoids and transcription.

The possible involvement of several transcription systems in the anticancer activity of carotenoids is the focus of this review. Carotenoids modulate the basic mechanisms of cell proliferation, growth factor signaling, gap junctional intercellular communication, and produce changes in the expression of many proteins participating in these processes. The changes in the expression of multiple proteins suggest that the initial effect of carotenoids involves modulation of transcription. We and others have found evidence for the role of several transcription systems, such as the retinoid receptors, activator protein-1 (AP-1), peroxisome proliferator-activated receptors (PPAR), xenobiotic receptors and the antioxidant response element (ARE), in the anticancer activity of carotenoids. The observed modulation of a network of transcription systems may provide the molecular basis for the synergistic anticancer effects of the combinations of various carotenoids together with other dietary and pharmacologic compounds.