Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains.

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here, we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals an additional mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains.

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction controls receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we identify key conserved residues and cancer-associated mutations that control CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells which can be modulated by disease-associated mutations or CTD phosphorylation. This work reveals a novel mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

Fisetin inhibits tau aggregation by interacting with the protein and preventing the formation of ß-strands.

Alzheimer's disease is a neurodegenerative disease which severely impacts the health of the elderly. Current treatments are only able to alleviate symptoms, but not prevent or cure the disease. The neurofibrillary tangles formed by tau protein aggregation are one of the defining characteristics of Alzheimer's disease, so tau protein has become a key target for the drug design. In this study, we show that fisetin, a plant-derived polyphenol compound, can inhibit aggregation of the tau fragment, K18, and can disaggregate tau K18 filaments in vitro. Meanwhile it is able to prevent the formation of tau aggregates in cells. Both experimental and computational studies indicate that fisetin could directly interact with tau K18 protein. The binding is mainly created by hydrogen bond and van der Waal force, prevents the formation of ß-strands at the two hexapeptide motifs, and does not perturb the secondary structure or the tubulin binding ability of tau protein. In summary, fisetin might be a candidate for further development as a potential preventive or therapeutic drug for Alzheimer's disease.

Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog.

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

Parkinson's Disease and Melanoma: Co-Occurrence and Mechanisms.

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta, depletion of dopamine in the striatum and the presence of Lewy bodies. Cancer is uncontrolled growth of cells in the body and migration of these cells from their site of origin to other parts of the body. PD and cancer are two opposite diseases, one arising from cell proliferation and the other from cell degeneration. This fundamental difference is consistent with inverse comorbidity between most cancers and neurodegenerative diseases. However, a positive association of PD and melanoma has been reported which has recently become of significant interest. A link between PD and cancer has been supported by many epidemiological studies, most of which show that PD patients have a lower risk of developing most cancers than the general population. However, the mechanisms underlying this epidemiological observation are not known. In this review we focus on epidemiological studies correlating PD and melanoma and the possible mechanisms underlying the co-occurrence of the two diseases. We explore possible explanations for the important observations that more PD patients develop melanoma that would otherwise be expected and vice-versa.

Phosphorylation regulates the secondary structure and function of dentin phosphoprotein peptides.

Dentin phosphoprotein (DPP) is the most acidic protein in vertebrates and structurally is classified as an intrinsically disordered protein. Functionally, DPP is related to dentin and bone formation, however the specifics of such association remain unknown. Here, we used atomistic molecular dynamics simulations to screen selected binding domains of DPP onto hydroxyapatite (HA), which is one of its important interacting partners. From these results, we selected a functionally relevant peptide, Ace-SSDSSDSSDSSDSSD-NH2 (named P5) and its phosphorylated form (named P5P), for experimental characterization. SAXS experiments indicated that in solution P5 was disordered, possibly in an extended conformation while P5P displayed more compact globular conformations. Circular dichroism and FTIR confirmed that, either in the presence or absence of Ca2+/HA, P5 adopts a random coil structure, whereas its phosphorylated counterpart, P5P, has a more compact arrangement associated with conformations that display ß-sheet and α-helix motifs when bound to HA. In solution, P5 inhibited HA crystal growth, whereas at similar concentrations, P5P stimulated it. These findings suggest that phosphorylation controls the transient formation of secondary and tertiary structure of DPP peptides, and, most likely of DPP itself, which in turn controls HA growth in solution and possibly HA growth in mineralized tissues.

The C-terminal α-helices of mammalian Hsc70 play a critical role in the stabilization of α-synuclein binding and inhibition of aggregation.

Protein misfolding, followed by aggregation and amyloid formation is an underlying pathological hallmark in a number of prevalent diseases, including Parkinson's (PD), Alzheimer's (AD) and Type 2 diabetes (T2D). In the case of PD, the aggregation of α-synuclein protein (α-syn) has been shown to be highly cytotoxic and to play a key role in the death of dopaminergic cells. Thus, inhibition of the aggregation process may be considered as an attractive avenue for therapeutic intervention. In this respect, molecular chaperones, known to promote proper folding of proteins, are able to inhibit protein aggregation thus preventing amyloid formation. In this work, the effect of the constitutively expressed chaperone Hsc70 and its various domains on α-syn aggregation have been investigated using different approaches. The results show that the C-terminal domain alone (residues 386-646) is as efficient in inhibiting α-syn aggregation as the entire Hsc70 protein, by increasing the lag phase for α-syn oligomeric nucleus formation, suggesting that the chaperone interacts with and stabilizes α-syn monomers and/or small aggregates. Deletion of the C-terminal helices (residues 510-646), which are known to play the role of a lid locking target peptide ligands in the peptide-binding site of the chaperone, strongly reduced the efficiency of inhibition of α-syn aggregation indicating that these helices play an essential in stabilizing the interaction between Hsc70 and α-syn. Furthermore, the effects of Hsc70 and its structural domains on aggregation appear to correlate with those on cytotoxicity, by reducing the fraction of α-syn toxic species to various degrees. Together these results suggest a mechanism in which inhibition of synuclein aggregation is the result of monomeric synuclein binding to the chaperone as any monomeric target unfolded protein or peptide binding to the chaperone.

Ginsenoside Rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils.

Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no ß-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.

The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of α-synuclein, and enhances its secretion and nuclear localization in cells.

A novel mutation in the α-Synuclein (α-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-Syn(G51D) behaves similarly to α-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregation.

c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease.

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD.

Assigning backbone NMR resonances for full length tau isoforms: efficient compromise between manual assignments and reduced dimensionality.

Tau protein is the longest disordered protein for which nearly complete backbone NMR resonance assignments have been reported. Full-length tau protein was initially assigned using a laborious combination of bootstrapping assignments from shorter tau fragments and conventional triple resonance NMR experiments. Subsequently it was reported that assignments of comparable quality could be obtained in a fully automated fashion from data obtained using reduced dimensionality NMR (RDNMR) experiments employing a large number of indirect dimensions. Although the latter strategy offers many advantages, it presents some difficulties if manual intervention, confirmation, or correction of the assignments is desirable, as may often be the case for long disordered and degenerate polypeptide sequences. Here we demonstrate that nearly complete backbone resonance assignments for full-length tau isoforms can be obtained without resorting either to bootstrapping from smaller fragments or to very high dimensionality experiments and automation. Instead, a set of RDNMR triple resonance experiments of modest dimensionality lend themselves readily to efficient and unambiguous manual assignments. An analysis of the backbone chemical shifts obtained in this fashion indicates several regions in full length tau with a notable propensity for helical or strand-like structure that are in good agreement with previous observations.

α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer.

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.

STARD4 abundance regulates sterol transport and sensing.

Nonvesicular transport of cholesterol plays an essential role in the distribution and regulation of cholesterol within cells, but it has been difficult to identify the key intracellular cholesterol transporters. The steroidogenic acute regulatory-related lipid-transfer (START) family of proteins is involved in several pathways of nonvesicular trafficking of sterols. Among them, STARD4 has been shown to increase intracellular cholesteryl ester formation and is controlled at the transcriptional level by sterol levels in cells. We found that STARD4 is very efficient in transporting sterol between membranes in vitro. Cholesterol levels are increased in STARD4-silenced cells, while sterol transport to the endocytic recycling compartment (ERC) and to the endoplasmic reticulum (ER) are enhanced upon STARD4 overexpression. STARD4 silencing attenuates cholesterol-mediated regulation of SREBP-2 activation, while its overexpression amplifies sterol sensing by SCAP/SREBP-2. To analyze STARD4's mode of action, we compared sterol transport mediated by STARD4 with that of a simple sterol carrier, methyl-ß-cyclodextrin (MCD), when STARD4 and MCD were overexpressed or injected into cells. Interestingly, STARD4 and cytosolic MCD act similarly by increasing the rate of transfer of sterol to the ERC and to the ER. Our results suggest that cholesterol transport mediated by STARD4 is an important component of the cholesterol homeostasis regulatory machinery.

Identification of a copper-binding metallothionein in pathogenic mycobacteria.

A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially binds up to six Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the gene's expression in Mtb up to 1,000-fold above normal expression. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionein (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function.

Structural effects of Parkinson's disease linked DJ-1 mutations.

Mutations in the protein DJ-1 are associated with familial forms of Parkinson's disease, indicating that DJ-1 may be involved in pathways related to the etiology of this disorder. Here we have used solution state NMR and circular dichroism spectroscopies to evaluate the extent of structural perturbations associated with five different Parkinson's disease linked DJ-1mutations: L166P, E64D, M26I, A104T, and D149A. Comparison of the data with those obtained for the wild-type protein shows that the L166P mutation leads to severe and global destabilization and unfolding of the protein structure, while the structure of the E64D mutation, as expected, is nearly unperturbed. Interestingly, the remaining three mutants all show different degrees of structural perturbation, which are accompanied by a reduction in the thermodynamic stability of the protein. The observed structural and thermodynamic differences are likely to underlie any functional variations between these mutants and the wild type, which in turn are likely responsible for the pathogenicity of these mutations.

Native chemical ligation in covalent caspase inhibition by p35.

Wide-spectrum caspase inhibition by the baculoviral p35 protein was previously shown to be a consequence of covalent inhibition in which a thioester bond is stably formed between the cleavage residue Asp87 of p35 and the active site Cys360' of caspase-8. Here we show that the N-terminal fragment of cleaved p35 (p35-N) is a circular peptide when dissociated from the caspase. Biochemical and crystallographic data suggest that p35-N circularization results from the trapping of a native chemical ligation intermediate in the p35/caspase complex, in which the N-terminal Cys2 of p35 attacks the Asp87-Cys360' thioester to form an equilibrium between Asp87-Cys2 and Asp87-Cys360'. This provides a crucial covalent interaction for keeping the N terminus of p35 bound in the caspase active site, which explains the absolute requirement of Cys2 for caspase inhibition. Participation of native chemical ligation in caspase inhibition by p35 illustrates an unusual mechanism of protease inhibition.

Residual structure in the repeat domain of tau: echoes of microtubule binding and paired helical filament formation.

The microtubule-associated protein tau is found aggregated into paired helical filaments in the intraneuronal neurofibrillary tangle deposits of victims of Alzheimer's disease (AD) and other related dementias. Tau contains a repeat domain consisting of three or four 31-32-residue imperfect repeats that forms the core of tau filaments and is capable of self-assembling into filaments in vitro. We have used high-resolution NMR spectroscopy to characterize the structural properties of the three-repeat domain of tau at the level of individual residues. We find that three distinct regions of the polypeptide corresponding to previously mapped microtubule interaction sites exhibit a preference for helical conformations, suggesting that these sites adopt a helical structure when bound to microtubules. In addition, we directly observe a marked preference for extended or beta-strand-like conformations in a stretch of residues between two of the helical regions, which corresponds closely to a region previously implicated as an early site of beta-strand structure formation and intermolecular interactions leading to paired helical filament (PHF) formation. This observation supports the idea that this region of the protein plays a crucial role in the formation of tau aggregates. We further show that disulfide-bond-mediated dimer formation does not affect and is not responsible for the observed structural preferences of the protein. Our results provide the first high-resolution view of the structural properties of the protein tau, are consistent with an important role for beta structure in PHF formation, and may also help explain recent reports that tau filaments contain helical structure.