Structure-Based Modeling of Sigma 1 Receptor Interactions with Ligands and Cholesterol and Implications for Its Biological Function.

The sigma 1 receptor (S1R) is a 223-amino-acid-long transmembrane endoplasmic reticulum (ER) protein. The S1R plays an important role in neuronal health and it is an established therapeutic target for neurodegenerative and neuropsychiatric disorders. Despite its importance in physiology and disease, the biological function of S1R is poorly understood. To gain insight into the biological and signaling functions of S1R, we took advantage of recently reported crystal structures of human and Xenopus S1Rs and performed structural modeling of S1R interactions with ligands and cholesterol in the presence of the membrane. By combining bioinformatics analysis of S1R sequence and structural modelling approaches, we proposed a model that suggests that S1R may exist in two distinct conformations-"dynamic monomer" (DM) and "anchored monomer" (AM). We further propose that equilibrium between AM and DM conformations of S1R is essential for its biological function in cells, with AM conformation facilitating the oligomerization of S1R and DM conformation facilitating deoligomerization. Consistent with experimental evidence, our hypothesis predicts that increased levels of membrane cholesterol and S1R antagonists should promote the oligomeric state of S1R, but S1R agonists and pathogenic mutations should promote its deoligomerization. Obtained results provide mechanistic insights into signaling functions of S1R in cells, and the proposed model may help to explain neuroprotective effects of S1R modulators.

Potential direct role of synuclein in dopamine transport and its implications for Parkinson's disease pathogenesis.

Parkinson Disease (PD) is a progressive neurodegenerative disorder that is caused by dysfunction and death of dopaminergic neurons. Mutations in the gene encoding α-synuclein (ASYN) have been linked with familial PD (FPD). Despite important role of ASYN in PD pathology, its normal biological function has not been clarified, although direct action of ASYN in synaptic transmission and dopamine (DA+) release have been proposed. In the present report we propose a novel hypothesis that ASYN functions as DA+/H+ exchanger that can facilitate transport of dopamine across synaptic vesicle (SV) membrane by taking advantage of proton gradient between SV lumen and cytoplasm. According to this hypothesis, normal physiological role of ASYN consists of fine-tuning levels of dopamine in the SVs based on cytosolic concentration of dopamine and intraluminal pH. This hypothesis is based on similarity in domain structure of ASYN and pHILP, a designed peptide developed to mediate loading of lipid nanoparticles with the cargo molecules. We reason that carboxy-terminal acidic loop D2b domain in both ASYN and pHILP binds cargo molecules. By mimicking DA+ association with E/D residues in D2b domain of ASYN using Tyrosine replacement approach (TR) we have been able to estimate that ASYN is able to transfer 8-12 molecules of dopamine across SV membrane on each DA+/H+ exchange cycle. Our results suggest that familial PD mutations (A30P, E46K, H50Q, G51D, A53T and A53E) will interfere with different steps of the exchange cycle, resulting in partial loss of dopamine transport function phenotype. We also predict that similar impairment in ASYN DA+/H+ exchange function also occurs as a result on neuronal aging due to changes in SV lipid composition and size and also dissipation of pH gradient across SV membrane. Proposed novel functional role of ASYN provides novel insights into its biological role and its role in PD pathogenesis.

Analysis of Non-Amyloidogenic Mutations in APP Supports Loss of Function Hypothesis of Alzheimer's Disease.

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in pathogenesis of Azheimer's disease (AD). Sequential cleavage of APP by ß- and γ-secretases leads to generation of Aß40 (non-amyloidogenic) and Aß42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) act as catalytic subunits of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 affect APP proteolysis by γ-secretase and influence levels of generated Aß40 and Aß42 peptides. The predominant idea in the field is the "amyloid hypothesis" that states that the resulting increase in Aß42:Aß40 ratio leads to "toxic gain of function" due to the accumulation of toxic Aß42 plaques and oligomers. An alternative hypothesis based on analysis of PS1 conditional knockout mice is that "loss of function" of γ-secretase plays an important role in AD pathogenesis. In the present paper, we propose a mechanistic hypothesis that may potentially reconcile these divergent ideas and observations. We propose that the presence of soluble Aß peptides in endosomal lumen (and secreted to the extracellular space) is essential for synaptic and neuronal function. Based on structural modeling of Aß peptides, we concluded that Aß42 peptides and Aß40 peptides containing non-amyloidogenic FAD mutations in APP have increased the energy of association with the membranes, resulting in reduced levels of soluble Aß in endosomal compartments. Analysis of PS1-FAD mutations also revealed that all of these mutations lead to significant reduction in both total levels of Aß produced and in the Aß40/Aß42 ratio, suggesting that the concentration of soluble Aß in the endosomal compartments is reduced as a result of these mutations. We further reasoned that similar changes in Aß production may also occur as a result of age-related accumulation of cholesterol and lipid oxidation products in postsynaptic spines. Our analysis more easily reconciled with the "loss of γ-secretase function" hypothesis than with the "toxic gain of Aß42 function" idea. These results may also explain why inhibitors of ß- and γ- secretase failed in clinical trials, as these compounds are also expected to significantly reduce soluble Aß levels in the endosomal compartments.

Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations.

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Sequential cleavage of APP by ß and γ secretases leads to the generation of Aß40 (non-amyloidogenic) and Aß42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aß42:Aß40 ratio and the accumulation of toxic Aß42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations-M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aß42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aß42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.

Differences in Recycling of Apolipoprotein E3 and E4-LDL Receptor Complexes-A Mechanistic Hypothesis.

Apolipoprotein E (ApoE) is a protein that plays an important role in the transport of fatty acids and cholesterol and in cellular signaling. On the surface of the cells, ApoE lipoparticles bind to low density lipoprotein receptors (LDLR) that mediate the uptake of the lipids and downstream signaling events. There are three alleles of the human ApoE gene. Presence of ApoE4 allele is a major risk factor for developing Alzheimer's disease (AD) and other disorders late in life, but the mechanisms responsible for biological differences between different ApoE isoforms are not well understood. We here propose that the differences between ApoE isoforms can be explained by differences in the pH-dependence of the association between ApoE3 and ApoE4 isoforms and LDL-A repeats of LDLR. As a result, the following endocytosis ApoE3-associated LDLRs are recycled back to the plasma membrane but ApoE4-containing LDLR complexes are trapped in late endosomes and targeted for degradation. The proposed mechanism is predicted to lead to a reduction in steady-state surface levels of LDLRs and impaired cellular signaling in ApoE4-expressing cells. We hope that this proposal will stimulate experimental research in this direction that allows the testing of our hypothesis.

Association with proteasome determines pathogenic threshold of polyglutamine expansion diseases.

Expansion of glutamine residue track (polyQ) within soluble protein is responsible for eight autosomal-dominant genetic neurodegenerative disorders. These disorders affect cerebellum, striatum, basal ganglia and other brain regions. Each disease develops when polyQ expansion exceeds a pathogenic threshold (Qth). A pathogenic threshold is unique for each disease but the reasons for variability in Qth within this family of proteins are poorly understood. In the previous publication we proposed that polarity of the regions flanking polyQ track in each protein plays a key role in defining Qth value [1]. To explain the correlation between the polarity of the flanking sequences and Qth we performed quantitative analysis of interactions between polyQ-expanded proteins and proteasome. Based on structural and theoretical modeling, we predict that Qth value is determined by the energy of polar interaction of the flanking regions with the polyQ and proteasome. More polar flanking regions facilitate unfolding of α-helical polyQ conformation adopted inside the proteasome and as a result, increase Qth. Predictions of our model are consistent with Qth values observed in clinic for each of the eight polyQ-expansion disorders. Our results suggest that the agents that can destabilize polyQ α-helical structure may have a beneficial therapeutic effect for treatment of polyQ-expansion disorders.

Crystal structure of the SH3 domain of growth factor receptor-bound protein 2.

This study presents the crystal structure of the N-terminal SH3 (SH3N) domain of growth factor receptor-bound protein 2 (Grb2) at 2.5 Šresolution. Grb2 is a small (215-amino-acid) adaptor protein that is widely expressed and involved in signal transduction/cell communication. The crystal structure of full-length Grb2 has previously been reported (PDB entry 1gri). The structure of the isolated SH3N domain is consistent with the full-length structure. The structure of the isolated SH3N domain was solved at a higher resolution (2.5 Šcompared with 3.1 Šfor the previously deposited structure) and made it possible to resolve some of the loops that were missing in the full-length structure. In addition, interactions between the carboxy-terminal region of the SH3N domain and the Sos1-binding sites were observed in the structure of the isolated domain. Analysis of these interactions provided new information about the ligand-binding properties of the SH3N domain of Grb2.

Reactions of Cyclometalated Platinum(II) [Pt(N∧C)(PR3)Cl] Complexes with Imidazole and Imidazole-Containing Biomolecules: Fine-Tuning of Reactivity and Photophysical Properties via Ligand Design.

This work describes interaction of a family of [Pt(N∧C)(PR3)Cl] complexes with imidazole (Im), possible application of this chemistry for regioselective labeling of proteins through imidazole rings of histidine residues and employment of the resulting phosphorescent products in bioimaging. It was found that the complexes containing aliphatic phosphines display reversible substitution of chloride ligand for imidazole function that required considerable excess of imidazole to obtain full conversion into the substituted [Pt(ppy)(PR3)(Im)] product, whereas the substitution in the complexes with aromatic phosphines readily proceeds in 1:1.5 mixture of reagents. Rapid, selective, and quantitative coordination of imidazole to the platinum complexes enabled regioselective labeling of ubiquitin. X-ray protein crystallography of the {[Pt(ppy)(PPh3)]/ubiquitin} conjugate revealed direct bonding of the platinum center to unique histidine-68 residue through the nitrogen atom of imidazole function, the coordination being also supported by noncovalent interaction of the ligands with the protein secondary structure. The variations of the cyclometalating N∧C ligands gave a series of [Pt(N∧C)(PPh3)Cl] complexes (N∧C = 2-phenylpyridine, 2-(benzofuran-3-yl)pyridine, 2-(benzo[b]thiophen-3-yl)pyridine, methyl-2-phenylquinoline-4-carboxylate), which were used to investigate the impact of N∧C-ligand onto photophysical properties of the imidazole complexes and conjugates with human serum albumin (HSA). The chloride ligand substitution for imidazole and formation of the conjugates results in ignition of the platinum chromophore luminescence with substantially higher quantum yield in the latter case. Variation of the metalating N∧C-ligand made possible the shift of the emission to the red region of visible spectrum for both types of the products. Cell-viability tests revealed low cytotoxicity of all {[Pt(N∧C)(PPh3)Cl]/HSA} conjugates, while PLIM experiments demonstrated their high potential for oxygen sensing.

The 2.2-Angstrom resolution crystal structure of the carboxy-terminal region of ataxin-3.

An expansion of polyglutamine (polyQ) sequence in ataxin-3 protein causes spinocerebellar ataxia type 3, an inherited neurodegenerative disorder. The crystal structure of the polyQ-containing carboxy-terminal fragment of human ataxin-3 was solved at 2.2-Å resolution. The Atxn3 carboxy-terminal fragment including 14 glutamine residues adopts both random coil and α-helical conformations in the crystal structure. The polyQ sequence in α-helical structure is stabilized by intrahelical hydrogen bonds mediated by glutamine side chains. The intrahelical hydrogen-bond interactions between glutamine side chains along the axis of the polyQ α-helix stabilize the secondary structure. Analysis of this structure furthers our understanding of the polyQ-structural characteristics that likely underlie the pathogenesis of polyQ-expansion disorders.

Pathogenic polyglutamine expansion length correlates with polarity of the flanking sequences.

BACKGROUND: Polyglutamine (polyQ) repeat expansion within coding sequence of a soluble protein is responsible for eight autosomal-dominant genetic neurodegenerative disorders. These disorders affect cerebellum, striatum, basal ganglia and other brain regions. The pathogenic polyQ-expansion threshold in these proteins varies from 32Q to 54Q. Understanding the reasons for variability in pathogenic polyQ threshold may provide insights into pathogenic mechanisms responsible for development of these disorders. FINDINGS: Here we established a quantitative correlation between the polarity of the flanking sequences and pathogenic polyQ-expansion threshold in this protein family. We introduced an "edge polarity index" (EPI) to quantify polarity effects of the flanking regions and established a strong correlation between EPI index and critical polyQ expansion length in this protein family. Based on this analysis we subdivided polyQ-expanded proteins into 2 groups - with strong and weak dependence of polyQ threshold on EPI index. The main difference between members of the first and the second group is a polarity profile of these proteins outside of polyQ and flanking regions. PolyQ proteins are known substrates for proteasome and most likely mechanistic explanation for the observed correlation is that proteasome may have an impaired ability to process continuous non-polar regions of proteins. CONCLUSIONS: The proposed hypothesis provides a quantitative explanation for variability in pathogenic threshold among polyQ-expansion disorders, which we established to correlate with polarity of flanking regions. To explain these results we propose that proteasome is not efficient in processing continuous non-polar regions of proteins, resulting in release of undigested and partially digested fragments. If supported experimentally, our hypothesis may have wide implications for further understanding the pathogensis of polyglutamine expansion disorders.

Beta conformation of polyglutamine track revealed by a crystal structure of Huntingtin N-terminal region with insertion of three histidine residues.

Huntington disease is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine (polyQ) expansion (> 35Q) in the first exon (EX1) of huntingtin protein (Htt). mHtt protein is thought to adopt one or more toxic conformation(s) that are involved in pathogenic interactions in cells . However, the structure of mHtt is not known. Here, we present a near atomic resolution structure of mHtt36Q-EX1. To facilitate crystallization, three histidine residues (3H) were introduced within the Htt36Q stretch resulting in the sequence of Q 7HQHQHQ 27. The Htt36Q3H region adopts α-helix, loop, ß-hairpin conformations. Furthermore, we observed interactions between the backbone of the Htt36Q3H ß-strand with the aromatic residues mimicking putative-toxic interactions with other proteins. Our findings support previous predictions that the expanded mHtt-polyQ region adopts a ß-sheet structure. Detailed structural information about mHtt improves our understanding of the pathogenic mechanisms in HD and other polyQ expansion disorders and may form the basis for rational design of small molecules that target toxic conformations of disease-causing proteins.