Direct evidence for GTP and GDP-inorganic phosphate intermediates in microtubule assembly

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100

The aggregation of ␣-synuclein (␣-Syn), the primary component of Lewy bodies, into high molecular weight assemblies is strongly associated with Parkinson disease. This event is believed to result from a conformational change within native ␣-Syn. Molecular chaperones exert critical housekeeping functions in vivo including refolding, maintaining in a soluble state, and/or pacifying protein aggregates. The influence of the stressinduced heat shock protein 70 (Hsp70) on ␣-Syn aggregation has been notably investigated. The constitutively expressed chaperone Hsc70 acts as an antiaggregation barrier before cells are overwhelmed with ␣-Syn aggregates and Hsp70 expression induced. Here, we investigate the interaction between Hsc70 and ␣-Syn, the consequences of this interaction, and the role of nucleotides and co-chaperones Hdj1 and Hdj2 as modulators. We show that Hsc70 sequesters soluble ␣-Syn in an assembly incompetent complex in the absence of ATP. The affinity of Hsc70 for soluble ␣-Syn diminishes upon addition of ATP alone or together with its co-chaperones Hdj1 or Hdj2 allowing faster binding and release of client proteins thus abolishing ␣-Syn assembly inhibition by Hsc70. We show that Hsc70 binds ␣-Syn fibrils with a 5-fold tighter affinity compared with soluble ␣-Syn. This suggests that Hsc70 preferentially interacts with high molecular weight ␣-Syn assemblies in vivo. Hsc70 binding certainly has an impact on the physicochemical properties of ␣-Syn assemblies. We show a reduced cellular toxicity of ␣-Syn fibrils coated with Hsc70 compared with "naked" fibrils. Hsc70 may therefore significantly affect the cellular propagation of ␣-Syn aggregates and their spread throughout the central nervous system in Parkinson disease.2 is one of the principal components of intracellular Lewy bodies, whose presence in the central nervous system is a defining feature of Parkinson disease (PD) and other synucleinopathies (1). Symptoms of PD are apparent after more than 70% of dopaminergic terminals and/or neurons have been lost, with some of the remaining neurons containing filamentous ␣-Syn. The precise cellular function of soluble ␣-Syn is unknown. There is, however, evidence that it plays an important role at presynaptic termini (2-4) and it has recently been suggested that the protein is a cellular ferrireductase (5). Various factors, including genetic susceptibility and environmental influences, can induce the aggregation of the naturally unfolded, soluble ␣-Syn (6, 7), leading to PD pathogenesis.Molecular chaperones assist newly synthesized proteins to reach their native-fold and are in charge of refolding misfolded or unfolded proteins (8). As the progression of PD is strongly associated with a change in ␣-Syn solubility, which is widely believed to be the consequence of a conformational change, there has been significant focus on the possible therapeutic role of molecular chaperones in the past 5 years (9). Previous investigations have focused on heat shock protein 70 (Hsp70) (10 -13), a molecular chaperone whose express...

“…Native luciferase activity was taken as 100%. The ATPase activity of Hsc70 alone or in the presence of its co-chaperones and unfolded luciferase was also monitored as described (24).…”

Section: Methodsmentioning

confidence: 99%

Hsc70 Protein Interaction with Soluble and Fibrillar α-Synuclein

Pemberton

Madiona

Pieri

et al. 2011

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100

“…GTPase Activity Measurements-GTP hydrolysis at 20°C was measured in assembly buffer containing 1 mM [␥- 32 P]GTP by extraction of the [ 32 P]phosphomolybdate complex formed in 1N HCl as described (30).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and Functional Analysis of the Assembly of Full-length Sup35p and Its Prion-forming Domain

Krzewska

Tanaka

Burston

et al. 2007

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Here we report a systematic comparison of the biochemical and assembly properties of full-length Sup35p and Sup35pNM. We show that the native structure of the C-terminal domain is retained within the fibrils. We determined the size of Sup35p nuclei and the critical concentration for assembly that both differ from that of Sup35pNM. We demonstrate that Sup35pNM co-assembles with the full-length protein and that fibrils made of Sup35p or Sup35pNM seed the assembly of soluble Sup35pNM and Sup35p with different efficiencies. Finally, we show that fibrils made of full-length Sup35p induce with higher efficiency [PSI ؉ ] appearance as compared with those made of Sup35pNM. Our findings reveal differences and similarities in the assembly of Sup35p and its NM fragment and validate the use of Sup35pNM in studying some aspects of Sup35p aggregation but also underline the importance of using full-length Sup35p in studying prion propagation both in vivo and in vitro.

“…ATPase Activity Measurements-ATP hydrolysis at 30°C was measured in folding buffer containing 2 mM [␥- 32 P]ATP and either 1 M CCT alone, CCT with denatured client proteins (0.1 mg/ml actin or ␤-tubulin), CCT with PhLP3 (0.9 mg/ml) or CCT with denatured client proteins and either full-length or truncated PhLP3 (tPhLP3 at 1.1 mg/ml), by extraction of the [ 32 P] phosphomolybdate complex formed in 1 N HCl as described (31).…”

Section: Methodsmentioning

confidence: 99%

PhLP3 Modulates CCT-mediated Actin and Tubulin Folding via Ternary Complexes with Substrates

Stirling

Cuéllar

Alfaro

et al. 2006

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105

Many ATP-dependent molecular chaperones, including Hsp70, Hsp90, and the chaperonins GroEL/Hsp60, require cofactor proteins to regulate their ATPase activities and thus folding functions in vivo. One conspicuous exception has been the eukaryotic chaperonin CCT, for which no regulator of its ATPase activity, other than non-native substrate proteins, is known. We identify the evolutionarily conserved PhLP3 (phosducin-like protein 3) as a modulator of CCT function in vitro and in vivo. PhLP3 binds CCT, spanning the cylindrical chaperonin cavity and contacting at least two subunits. When present in a ternary complex with CCT and an actin or tubulin substrate, PhLP3 significantly diminishes the chaperonin ATPase activity, and accordingly, excess PhLP3 perturbs actin or tubulin folding in vitro. Most interestingly, however, the Saccharomyces cerevisiae PhLP3 homologue is required for proper actin and tubulin function. This cellular role of PhLP3 is most apparent in a strain that also lacks prefoldin, a chaperone that facilitates CCT-mediated actin and tubulin folding. We propose that the antagonistic actions of PhLP3 and prefoldin serve to modulate CCT activity and play a key role in establishing a functional cytoskeleton in vivo.