Alla Shainskaya

Monoubiquitylation of α-Synuclein by Seven in Absentia Homolog (SIAH) Promotes Its Aggregation in Dopaminergic Cells

α-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of α-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. α-Synuclein is ubiquitylated in Lewy bodies, but the role of α-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates α-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates α-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished α-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates α-synuclein. Moreover, SIAH co-immunoprecipitated with α-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of α-synuclein by SIAH. Monoubiquitylation increased the aggregation of α-synuclein in vitro. At the electron microscopy level, monoubiquitylated α-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased α-synuclein aggregation in vivo as observed by increased formation of α-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for α-synuclein aggregation and Lewy body formation.

Renal Na,K-ATPase in Genetic Hypertension

Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb+ occlusion, turnover number, alpha 1- and beta 1-subunit protein abundance, and alpha 1 and beta 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb+ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of alpha 1 and beta 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.

Mitotic Phosphorylation of the Peripheral Golgi Protein Nir2 by Cdk1 Provides a Docking Mechanism for Plk1 and Affects Cytokinesis Completion

The rearrangement of the Golgi apparatus during mitosis is regulated by several protein kinases, including Cdk1 and Plk1. Several peripheral Golgi proteins that dissociate from the Golgi during mitosis are implicated in regulation of cytokinesis or chromosome segregation, thereby coordinating mitotic and cytokinetic events to Golgi rearrangement. Here we show that, at the onset of mitosis, Cdk1 phosphorylates the peripheral Golgi protein Nir2 at multiple sites; of these, S382 is the most prominent. Phosphorylation of Nir2 by Cdk1 facilitates its dissociation from the Golgi apparatus, and phospho-Nir2(pS382) is localized in the cleavage furrow and midbody during cytokinesis. Mitotic phosphorylation of Nir2 is required for docking of the phospho-Ser/Thr binding module, the Polo box domain of Plk1, and overexpression of a Nir2 mutant, which fails to interact with Plk1, affects the completion of cytokinesis. These results demonstrate a mechanism for coordinating mitotic and cytokinetic events with Golgi rearrangement during cell division.

Distribution and function of new bacterial intein-like protein domains.

Hint protein domains appear in inteins and in the C‐terminal region of Hedgehog and Hedgehog‐like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein‐splicing of their host‐protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N‐terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein‐like (BIL) domains. Bacterial intein‐like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C‐terminal flank and could also protein‐splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

Acetylation represses the binding of CheY to its target proteins

The ability of CheY, the response regulator of bacterial chemotaxis, to generate clockwise rotation is regulated by two covalent modifications – phosphorylation and acetylation. While the function and signal propagation of the former are widely understood, the mechanism and role of the latter are still obscure. To obtain information on the function of this acetylation, we non‐enzymatically acetylated CheY to a level similar to that found in vivo, and examined its binding to its kinase CheA, its phosphatase CheZ and the switch protein FliM – its target at the flagellar switch complex. Acetylation repressed the binding to all three proteins. These results suggest that both phosphorylation and acetylation determine CheYs ability to bind to its target proteins, thus providing two levels of regulation, fast and slow respectively. The fast level is modulated by environmental signals (e.g. chemotactic and thermotactic stimuli). The slow one is regulated by the metabolic state of the cell and it determines, at each metabolic state, the fraction of CheY molecules that can participate in signalling.

Selective Serotonin Reuptake Inhibitors (SSRIs) Inhibit Insulin Secretion and Action in Pancreatic β Cells

Background: Selective serotonin reuptake inhibitors (SSRIs) are used for the treatment of mood and anxiety disorders. Results: SSRIs inhibit insulin action and secretion, promote the unfolded protein response, and induce apoptosis of pancreatic β cells. Conclusion: SSRIs inhibit insulin signaling and beta cell function. Significance: SSRIs might accelerate the transition from an insulin-resistant state to overt diabetes. Selective serotonin reuptake inhibitors (SSRIs) are antidepressants used for the treatment of mood and anxiety disorders. Here, we demonstrate that incubation (2 h) of murine islets or Min6 β cell line with the SSRIs paroxetine, fluoxetine, or sertraline inhibited insulin-induced Tyr phosphorylation of insulin receptor substrate (IRS)-2 protein and the activation of its downstream targets Akt and the ribosomal protein S6 kinase-1 (S6K1). Inhibition was dose-dependent with half-maximal effects at ∼15–20 μm. It correlated with a rapid dephosphorylation and activation of the IRS kinase GSK3β. Introduction of GSK3β siRNAs eliminated the inhibitory effects of the SSRIs. Inhibition of IRS-2 action by 30 μm SSRI was associated with a marked inhibition of glucose-stimulated insulin secretion from murine and human pancreatic islets. Secretion induced by basic secretagogues (KCl and Arg) was not affected by these drugs. Prolonged treatment (16 h) of Min6 cells with sertraline resulted in the induction of inducible nitric oxide synthase; activation of endoplasmic reticulum stress, and the initiation of the unfolded protein response, manifested by enhanced transcription of ATF4 and C/EBP homologous protein. This triggered an apoptotic process, manifested by enhanced caspase 3/7 activity, which resulted in β cell death. These findings implicate SSRIs as inhibitors of IRS protein function and insulin action through the activation of GSK3β. They further suggest that SSRIs inhibit insulin secretion; induce the unfolded protein response; activate an apoptotic process, and trigger β cell death. Given that SSRIs promote insulin resistance while inhibiting insulin secretion, these drugs might accelerate the transition from an insulin-resistant state to overt diabetes.

In Vivo Acetylation of CheY, a Response Regulator in Chemotaxis of Escherichia coli

CheY, the excitatory response regulator in the chemotaxis system of Escherichia coli, can be modulated by two covalent modifications: phosphorylation and acetylation. Both modifications have been detected in vitro only. The role of CheY acetylation is still obscure, although it is known to be involved in chemotaxis and to occur in vitro by two mechanisms--acetyl-CoA synthetase-catalyzed transfer of acetyl groups from acetate to CheY and autocatalyzed transfer from AcCoA. Here, we succeeded in detecting CheY acetylation in vivo by three means--Western blotting with a specific anti-acetyl-lysine antibody, mass spectrometry, and radiolabeling with [(14)C]acetate in the presence of protein-synthesis inhibitor. Unexpectedly, the level and rate of CheY acetylation in vivo were much higher than that in vitro. Thus, before any treatment, 9-13% of the lysine residues were found acetylated, depending on the growth phase, meaning that, on average, essentially every CheY molecule was acetylated in vivo. This high level was mainly the outcome of autoacetylation. Addition of acetate caused an incremental increase in the acetylation level, in which acetyl-CoA synthetase was involved too. These findings may have far-reaching implications for the structure-function relationship of CheY.

Calcium-mediated interactions regulate the subcellular localization of extracellular signal-regulated kinases.

The subcellular localization of ERKs in cells, which is important for proper signaling, may be regulated through protein-protein interactions. We found that inactive ERK2 interacts with a large number of proteins through its cytosolic retention sequence/common docking domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERK interactions also influenced the localization of ERKs, as calcium chelators enhanced nuclear translocation, whereas elevated calcium levels prevented it. This effect of calcium was apparent upon lysophosphatidic acid stimulation, where ERKs translocation was delayed compared with that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERK translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. Upon stimulation, ERKs are phosphorylated and released from cytoplasmic anchors to allow shuttling toward the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and, therefore, also their spatiotemporal regulation. Thus, calcium regulates ERK localization, which is important for the compartmentalization of ERKs with their proper substrates and thereby their signaling specificity.

Acid Beta-Glucosidase: Insights from Structural Analysis and Relevance to Gaucher Disease Therapy.

Abstract In mammalian cells, glucosylceramide (GlcCer), the simplest glycosphingolipid, is hydrolyzed by the lysosomal enzyme acid β-glucosidase (GlcCerase). In the human metabolic disorder Gaucher disease, GlcCerase activity is significantly decreased owing to one of approximately 200 mutations in the GlcCerase gene. The most common therapy for Gaucher disease is enzyme replacement therapy (ERT), in which patients are given intravenous injections of recombinant human GlcCerase; the Genzyme product Cerezyme® has been used clinically for more than 15 years and is administered to approximately 4000 patients worldwide. Here we review the crystal structure of Cerezyme® and other recombinant forms of GlcCerase, as well as of their complexes with covalent and non-covalent inhibitors. We also discuss the stability of Cerezyme®, which can be altered by modification of its N-glycan chains with possible implications for improved ERT in Gaucher disease.

Fe2+-catalyzed Oxidative Cleavages of Ca2+-ATPase Reveal Novel Features of Its Pumping Mechanism

We have analyzed the Fe2+-catalyzed oxidative cleavages of Ca2+-ATPase in the presence of Ca2+, with or without the ATP analog 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+-ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+-ATPase and H+,K+-ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg·ATP stabilizes the N to P interaction (E1·Mg·ATP conformation).