Stimulation of Ca2+-ATPase Transport Activity by a Small-Molecule Drug

The sarco(endo)plasmic reticulum Ca(2+)−ATPase (SERCA) hydrolyzes ATP to transport Ca(2+) from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small‐molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP‐dependent Ca(2+) translocation. The concentration dependence of the CDN1163 effect provided an EC(50)=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca(2+) concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca(2+) release into the SR lumen

Synthesis of Functionalized 2-(4-Hydroxyphenyl)-3-methylbenzofuran Allosteric Modulators of Hsp90 Activity

Hsp90 is a molecular chaperone that plays a pivotal role in the cell life cycle. ATP-regulated internal dynamics are critical to Hsp90 function and we recently demonstrated that these dynamics can be modulated in an allosteric fashion; the protein C-terminal domain (CTD) can be effectively targeted with a family of 2-phenyl-benzofuran derivatives. Here we describe the expansion of the initial library, reporting 28 new derivatives that explore the chemical space at opposite ends of the benzofuran scaffold. Interactions of the compounds with a full-length protein homolog were explored by Saturation Transfer Difference (STD) NMR spectroscopy. In this context we also report the interaction epitope of Novobiocin, a known CTD inhibitor

A New Surface Plasmon Resonance Assay for in Vitro Screening of Mannose-Binding Lectin Inhibitors

Mannose-binding lectin (MBL) is a circulating protein that acts as a soluble pattern recognition molecule of the innate immunity. It binds to carbohydrate patterns on the surface of pathogens or of altered self-cells, with activation of the lectin pathway of the complement system. Recent evidence indicates that MBL contributes to the pathophysiology of ischemia-reperfusion injury and other conditions. Thus, MBL inhibitors offer promising therapeutic strategies, since they prevent the interaction of MBL with its target sugar arrays. We developed and characterized a novel assay based on surface plasmon resonance for in vitro screening of these compounds, which may be useful before the more expensive and time-consuming in vivo studies. The assay measures the inhibitor's ability to interfere with the binding of murine MBL-A or MBL-C, or of human recombinant MBL, to mannose residues immobilized on the sensor chip surface. We have applied the assay to measure the IC50 of synthetic glycodendrimers, two of them with neuroprotective properties in animal models of MBL-mediated injuries

Splice Isoforms of the Polyglutamine Disease Protein Ataxin-3 Exhibit Similar Enzymatic yet Different Aggregation Properties

Protein context clearly influences neurotoxicity in polyglutamine diseases, but the contribution of alternative splicing to this phenomenon has rarely been investigated. Ataxin-3, a deubiquitinating enzyme and the disease protein in SCA3, is alternatively spliced to encode either a C-terminal hydrophobic stretch or a third ubiquitin interacting motif (termed 2UIM and 3UIM isoforms, respectively). In light of emerging insights into ataxin-3 function, we examined the significance of this splice variation. We confirmed neural expression of several minor 5′ variants and both of the known 3′ ataxin-3 splice variants. Regardless of polyglutamine expansion, 3UIM ataxin-3 is the predominant isoform in brain. Although 2UIM and 3UIM ataxin-3 display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and more rapidly degraded by the proteasome. Our data demonstrate how alternative splicing of sequences distinct from the trinucleotide repeat can alter properties of the encoded polyglutamine disease protein and thereby perhaps contribute to selective neurotoxicity

Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways

The risk of developing neurodegenerative diseases increases with age. Although many of the molecular pathways regulating proteotoxic stress and longevity are well characterized, their contribution to disease susceptibility remains unclear. In this study, we describe a new Caenorhabditis elegans model of Machado–Joseph disease pathogenesis. Pan-neuronal expression of mutant ATXN3 leads to a polyQ-length dependent, neuron subtype-specific aggregation and neuronal dysfunction. Analysis of different neurons revealed a pattern of dorsal nerve cord and sensory neuron susceptibility to mutant ataxin-3 that was distinct from the aggregation and toxicity profiles of polyQ-alone proteins. This reveals that the sequences flanking the polyQ-stretch in ATXN3 have a dominant influence on cell-intrinsic neuronal factors that modulate polyQ-mediated athogenesis. Aging influences the ATXN3 phenotypes which can be suppressed by the nregulation of the insulin/insulin growth factor-1-like signaling pathway and activation of heat shock factor-1.This work was supported by grants from Fundacão Ciência eTecnologia (FCT) to P.M. (PTDC/SAU-GMG/64076/2006, PTDC/SAU-GMG/112617/2009, SFRH/BD/27258/2006 to A.T.C., UMINHO/BI/052/2010 to A.J. and SFRH/BD/51059/2010 to A.N.C.), from the National Ataxia Foundation to PM and from the National Institutes of Health (NIGMS, NIA and NINDS) to R.M. This work was also granted by the Hospital San Rafael (Coruna) with the Rafael Hervada prize on Biomedical Research (2010)

Understanding the Role of the Josephin Domain in the PolyUb Binding and Cleavage Properties of Ataxin-3

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology