Igor Tsigelny

The Arg451Cys-Neuroligin-3 Mutation Associated with Autism Reveals a Defect in Protein Processing

The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the β-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished β-neurexin-1 (NX1β) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1β. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.

Cell-to-cell transmission of non-prion protein aggregates

Neurodegenerative disorders such as Alzheimer disease, Parkinson disease, frontotemporal dementia, Huntington disease and Creutzfeldt–Jakob disease (CJD) are characterized by progressive accumulation of protein aggregates in selected brain regions. Protein misfolding and templated assembly into aggregates might result from an imbalance between protein synthesis, aggregation and clearance. Although protein misfolding and aggregation occur in most neurodegenerative disorders, the concept of spreading and infectivity of aggregates in the CNS has, until now, been confined to prion diseases such as CJD and bovine spongiform encephalopathy. Emerging evidence, however, suggests that prion-like spreading, involving secreted proteins such as amyloid-β and cytosolic proteins such as tau, huntingtin and α-synuclein, can occur in other neurodegenerative disorders. The underlying molecular mechanisms and the therapeutic implications of the new data are discussed in this article.

Mechanisms of Hybrid Oligomer Formation in the Pathogenesis of Combined Alzheimer's and Parkinson's Diseases

Background Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimers disease (AD) and Parkinsons disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear. Methodology/Principal Findings In order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels. Conclusion/Significance These results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

Dynamics of α-synuclein aggregation and inhibition of pore-like oligomer development by β-synuclein

Accumulation of α‐synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinsons disease and Lewy body dementia. In contrast, β‐synuclein (β‐syn), a close homologue, does not aggregate and reduces α‐synuclein (α‐syn)‐related pathology. Although considerable information is available about the conformation of α‐syn at the initial and end stages of fibrillation, less is known about the dynamic process of α‐syn conversion to oligomers and how interactions with antiaggregation chaperones such as β‐synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle‐derived structure of α‐syn showed that α‐syn homodimers can adopt nonpropagating (head‐to‐tail) and propagating (head‐to‐head) conformations. Propagating α‐syn dimers on the membrane incorporate additional α‐syn molecules, leading to the formation of pentamers and hexamers forming a ring‐like structure. In contrast, β‐syn dimers do not propagate and block the aggregation of α‐syn into ring‐like oligomers. Under in vitro cell‐free conditions, α‐syn aggregates formed ring‐like structures that were disrupted by β‐syn. Similarly, cells expressing α‐syn displayed increased ion current activity consistent with the formation of Zn2+‐sensitive nonselective cation channels. These results support the contention that in Parkinsons disease and Lewy body dementia, α‐syn oligomers on the membrane might form pore‐like structures, and that the beneficial effects of β‐synuclein might be related to its ability to block the formation of pore‐like structures.

Role of the Glycine Triad in the ATP-binding Site of cAMP-dependent Protein Kinase*

A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed inEscherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on bothK m and k cat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5–8-fold reduction in K m  (ATP), an 8–12-fold increase in K m  (peptide), and a 3–5-fold drop in k cat. TheK m  (ATP) andK m  (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and thek cat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.

Tryptophan Fluorescence Reveals Conformational Changes in the Acetylcholine Binding Protein

The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the α-neurotoxins. Since agonist binding occurs in millisecond time frames, and the α-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.

Fibrinogen Signal Transduction as a Mediator and Therapeutic Target in Inflammation:Lessons from Multiple Sclerosis

The blood protein fibrinogen as a ligand for integrin and non-integrin receptors functions as the molecular nexus of coagulation, inflammation and immunity. Studies in animal models and in human disease have demonstrated that extravascular fibrinogen that is deposited in tissues upon vascular rupture is not merely a marker, but a mediator of diseases with an inflammatory component, such as rheumatoid arthritis, multiple sclerosis, sepsis, myocardial infarction and bacterial infection. The present article focuses on the recent discoveries of specific cellular targets and receptors for fibrinogen within tissues that have extended the role of fibrinogen from a coagulation factor to a regulator of inflammation and immunity. Fibrinogen has the potential for selective drug targeting that would target its proinflammatory properties without affecting its beneficial effects in hemostasis, since it interacts with different receptors to mediate blood coagulation and inflammation. Strategies to target receptors for fibrinogen and fibrin within the tissue microenvironment could reveal selective and disease-specific agents for therapeutic intervention in a variety of human diseases associated with fibrin deposition.

Untargeted metabolomics identifies enterobiome metabolites and putative uremic toxins as substrates of organic anion transporter 1 (Oat1).

Untargeted metabolomics on the plasma and urine from wild-type and organic anion transporter-1 (Oat1/Slc22a6) knockout mice identified a number of physiologically important metabolites, including several not previously linked to Oat1-mediated transport. Several, such as indoxyl sulfate, derive from Phase II metabolism of enteric gut precursors and accumulate in chronic kidney disease (CKD). Other compounds included vitamins (pantothenic acid, 4-pyridoxic acid), urate, and metabolites in the tryptophan and nucleoside pathways. Three metabolites, indoxyl sulfate, kynurenine, and xanthurenic acid, were elevated in the plasma and interacted strongly and directly with Oat1 in vitro with IC50 of 18, 12, and 50 μM, respectively. A pharmacophore model based on several identified Oat1 substrates was used to screen the NCI database and candidate compounds interacting with Oat1 were validated in an in vitro assay. Together, the data suggest a complex, previously unidentified remote communication between the gut microbiome, Phase II metabolism in the liver, and elimination via Oats of the kidney, as well as indicating the importance of Oat1 in the handling of endogenous toxins associated with renal failure and uremia. The possibility that some of the compounds identified may be part of a larger remote sensing and signaling pathway is also discussed.

Identification of the protein kinase A regulatory RIα-catalytic subunit interface by amide H/2H exchange and protein docking

An important goal after structural genomics is to build up the structures of higher-order protein–protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and RIα regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen/deuterium (H/2H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A- and B-helices of RIα is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.

Role of α-synuclein penetration into the membrane in the mechanisms of oligomer pore formation.

Parkinson’s disease (PD) and dementia with Lewy bodies are common disorders of the aging population and characterized by the progressive accumulation of α‐synuclein (α‐syn) in the central nervous system. Aggregation of α‐syn into oligomers with a ring‐like appearance has been proposed to play a role in toxicity. However, the molecular mechanisms and the potential sequence of events involved in the formation of pore‐like structures are unclear. We utilized computer modeling and cell‐based studies to investigate the process of oligomerization of wild‐type and A53T mutant α‐syn in membranes. The studies suggest that α‐syn penetrates the membrane rapidly, changing its conformation from α‐helical towards a coiled structure. This penetration facilitates the incorporation of additional α‐syn monomers in the complex, and the subsequent displacement of phospholipids and the formation of oligomers in the membrane. This process occurred more rapidly, and with a more favorable energy of interaction, for mutant A53T compared with wild‐type α‐syn. After 4 ns of simulation of the protein–membrane model, α‐syn had penetrated through two‐thirds of the membrane. By 9 ns, the penetration of the annular α‐syn oligomers can result in the formation of pore‐like structures that fully perforate the lipid bilayer. Experimental incubation of recombinant α‐syn in synthetic membranes resulted in the formation of similar pore‐like complexes. Moreover, mutant (A53T) α‐syn had a greater tendency to accumulate in neuronal membrane fractions in cell cultures, resulting in greater neuronal permeability, as demonstrated with the calcein efflux assay. These studies provide a sequential molecular explanation for the process of α‐syn oligomerization in the membrane, and support the role of formation of pore‐like structures in the pathogenesis of the neurodegenerative process in PD.