Shubhik DebBurman

alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress.

Parkinsons disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.Parkinsons disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein α-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type α-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T α-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, α-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed α-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of α-synuclein. Both sen3-1 and doa3-1 mutants exhibited synthetic lethality with α-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), α-synuclein was extremely lethal to cells that lacked managanese superoxide dismutase Mn-SOD (sod2Δ) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Δ). Despite the toxicity, sod2Δ cells never displayed intracellular aggregates of α-synuclein. We suggest that the toxic α-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve α-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate α-synuclein toxicity.

Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors.

Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.

α-synuclein fission yeast model : Concentration-dependent aggregation without plasma membrane localization or toxicity

Despite fission yeasts history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because α-synuclein misfolding and aggregation are linked to Parkinsons disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates α-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type α-synuclein and three mutants (A 30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for α-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T α-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dipendent manner, whereas A30P and A30P/A53T remained diffuse throughhout the cytoplasm. A53T α-synuclein for med aggregates faster than wild-type α-synuclein and at a lower α-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T α-synuclein did not target to the plasma membrane in fission yeast, not even at low α-synuclein concentrations or as a precursor step to forming aggregates. Despite α-synucleins extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that α-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of α-synuclein biology, and both organisms shed insight into α-synucleins role in PD pathogenesis.

Chapter 15 The role of G-protein coupled receptor kinases in the regulation of muscarinic cholinergic receptors

Publisher Summary This chapter focuses exclusively on molecular events underlying homologous desensitization of muscarinic acetylcholine receptors (mAChR) by a unique family of protein kinases that appear to specifically recognize GPRs. Other protein kinases, such as protein kinase C, may also be involved in mAChR desensitization. The term “desensitization”, when applied to the GPRs, actually refers to a collection of events that may be independently regulated, and not necessarily sequential. The hallmarks of desensitization of the GPRs include uncoupling of the receptors from G-proteins and/or loss of high affinity agonist binding. These events occur rapidly and appear to involve phosphorylation of the GPRs by specific and/or generic protein kinases. This phosphorylation induced uncoupling is proposed to play a physiological role in terminating signalling in the sensory receptor systems. The chapter identifies the kinases that phosphorylate the mAChRs in vivo employs a system, whereby it attempts to take apart the system and reconstitutes agonist-dependent phosphorylation and desensitization of the receptors in vitro . For this system, the chapter purifies chick heart or recombinant human m2 mAChRs and reconstitutes the receptors into phospholipid vesicles.

Contribution of Alanine-76 and Serine Phosphorylation in α-Synuclein Membrane Association and Aggregation in Yeasts

In Parkinsons disease (PD), misfolded and aggregated α-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 in α-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions to α-synuclein membrane association are less clear. We found that α-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessing α-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) altered α-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreased α-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions to α-synuclein aggregation and are the first to detail their impact on α-synucleins plasma membrane and endomembrane association.

Familial Parkinson's Disease Mutant E46K α-Synuclein Localizes to Membranous Structures, Forms Aggregates, and Induces Toxicity in Yeast Models

In Parkinsons disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells.