Conformational properties of the aggregation precursor state of HypF-N

Campioni, Silvia; Mossuto, Maria F.; Torrassa, Silvia; Calloni, Giulia; Laureto, Patrizia Polverino de; Relini, Annalisa; Fontana, Angelo; Chiti, Fabrizio

doi:10.1016/j.jmb.2008.04.002

Cited by 44 publications

(86 citation statements)

References 64 publications

(85 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…HypF-N was expressed in E. coli cells and purified using an affinity chromatography column packed with the HIS-Select Nickel Affinity Gel, as previously described (Campioni et al, 2008). …”

Section: Formation Of Hypf-n Toxic Oligomersmentioning

confidence: 99%

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Cappelli

Penco²,

Mannini³

et al. 2016

Biological Chemistry

Self Cite

View full text Add to dashboard Cite

. (2016). Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations. Biological Chemistry: official scientific journal of the GBM, 397 (5), 401-415. Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations AbstractLiving systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication DetailsCappelli, S., Penco, A., Mannini, B., Cascella, R., Wilson, M. R., Ecroyd, H., Li, X., Buxbaum, J. N., Dobson, C. M., Cecchi, C., Relini, A. & Chiti, F. (2016 measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasises the efficiency and versatility of these protein molecules.

show abstract

“…HypF-N was expressed in E. coli cells and purified using an affinity chromatography column packed with the HIS-Select Nickel Affinity Gel, as previously described (Campioni et al, 2008). …”

Section: Formation Of Hypf-n Toxic Oligomersmentioning

confidence: 99%

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Cappelli

Penco²,

Mannini³

et al. 2016

Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…In the present study we performed a proteomic analysis of NIH-3T3 cells exposed to toxic aggregates of a protein domain not involved in any amyloid disease, the N-terminal domain of the prokaryotic HypF hydrogenase maturation factor (HypF-N) whose aggregation properties and aggregate cytotoxicity were previously characterized [8,10,17]. To our knowledge, this is the first proteomic study on the alterations of the protein expression profiles in cells exposed to toxic amyloid aggregates of a disease-unrelated protein.…”

Section: Biochimica Et Biophysica Acta 1794 (2009) 1243-1250mentioning

confidence: 99%

“…HypF-N was expressed and purified as previously described [17]. HypF-N prefibrillar aggregates were obtained by incubating the protein for 48 h at room temperature at a concentration of 0.3 mg/ ml in 30% (v/v) trifluoroethanol, 50 mM sodium acetate, 2 mM dithiotreitol (DTT), pH 5.5, as previously reported [17].…”

Section: Hypf-n Expression and Purificationmentioning

confidence: 99%

“…HypF-N prefibrillar aggregates were obtained by incubating the protein for 48 h at room temperature at a concentration of 0.3 mg/ ml in 30% (v/v) trifluoroethanol, 50 mM sodium acetate, 2 mM dithiotreitol (DTT), pH 5.5, as previously reported [17]. At the end of the incubation the solution was centrifuged, and the resulting pellet was dried under N 2 to remove the residual solvent, dissolved in DMEM at 200 μM (monomeric protein concentration) and immediately added to the cell culture medium at 2 μM final concentration.…”

Section: Hypf-n Expression and Purificationmentioning

confidence: 99%

See 1 more Smart Citation

Proteomic analysis of cells exposed to prefibrillar aggregates of HypF-N

Magherini

Pieri

Guidi

et al. 2009

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

View full text Add to dashboard Cite

Several human diseases are associated with the deposition of stable ordered protein aggregates known as amyloid fibrils. In addition, a large wealth of data shows that proteins not involved in amyloidoses, are able to form, in vitro, amyloid-like prefibrillar and fibrillar assemblies indistinguishable from those grown from proteins associated with disease. Previous studies showed that early prefibrillar aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) are cytotoxic, inducing early mitochondria membrane depolarization, activation of caspase 9 and eventually cell death. To gain knowledge on the molecular basis of HypF-N aggregate cytotoxicity, we performed a differential proteomic analysis of NIH-3T3 cells exposed to HypF-N prefibrillar aggregates in comparison with control cells. Two-dimensional gel electrophoresis followed by protein identification by MALDI-TOF MS, allowed us to identify 21 proteins differentially expressed. The changes of the expression level of proteins involved in stress response (Hsp60 and 78 kDa glucose-regulated protein) and in signal transduction (Focal adhesion kinase1) appear particularly interesting as possible determinants of the cell fate. The levels of some of the differently expressed proteins were modified also in similar studies carried out on cells exposed to Aβ or α-synuclein aggregates, supporting the existence of shared features of amyloid cytotoxicity.

show abstract

“…Chaperonins are specialized in binding to partially unfolded intermediates, often of molten-globule type possessing significant structure. Such intermediates are notorious for their specific aggregation prone behavior [5,6]. One important role of a molecular chaperone is to provide a container that prevents intermolecular interactions and keeps the substrate protein secluded from bulk proteins.…”

Section: Introductionmentioning

confidence: 99%

GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study

et al. 2010

View full text Add to dashboard Cite

The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single-and double-cysteine mutants were specifically spinlabeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20°C (as close as ∼8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin-spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60°C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.

show abstract

Conformational properties of the aggregation precursor state of HypF-N

Cited by 44 publications

References 64 publications

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Proteomic analysis of cells exposed to prefibrillar aggregates of HypF-N

GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study

Contact Info

Product

Resources

About