Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Oct 19;430(21):4195-4208.
doi: 10.1016/j.jmb.2018.08.016. Epub 2018 Aug 18.

Polyphosphate Stabilizes Protein Unfolding Intermediates as Soluble Amyloid-like Oligomers

Affiliations
Free PMC article

Polyphosphate Stabilizes Protein Unfolding Intermediates as Soluble Amyloid-like Oligomers

Nicholas G Yoo et al. J Mol Biol. .
Free PMC article

Abstract

Inorganic polyphosphate (polyP) constitutes one of the most conserved and ubiquitous molecules in biology. Recent work in bacteria demonstrated that polyP increases oxidative stress resistance by preventing stress-induced protein aggregation and promotes biofilm formation by stimulating functional amyloid formation. To gain insights into these two seemingly contradictory functions of polyP, we investigated the effects of polyP on the folding model lactate dehydrogenase. We discovered that the presence of polyP during the thermal unfolding process stabilizes folding intermediates of lactate dehydrogenase as soluble micro-β-aggregates with amyloid-like properties. Size and heterogeneity of the oligomers formed in this process were dependent on the polyP chain length, with longer chains forming smaller, more homogenous complexes. This ability of polyP to stabilize thermally unfolded proteins even upon exposure to extreme temperatures appears to contribute to the observed resistance of uropathogenic Escherichia coli toward severe heat shock treatment. These results suggest that the working mechanism of polyP is the same for both soluble and amyloidogenic proteins, with the ultimate outcome likely being determined by a combination of polyP chain length and the client protein itself. They help to explain how polyP can simultaneously function as general stress-protective chaperone and instigator of amyloidogenic processes in vivo.

Keywords: Polyphosphate; amyloid-like aggregates; chaperone; heat shock; protein unfolding.

Figures

Figure 1.
Figure 1.. PolyP stabilizes thermally unfolded intermediates of LDH
(A) LDH (5.7 μM) was heated in the spectrophotometer in the absence (black) or in the presence of 1 mM polyP14 (green), polyP130 (red), or polyP300 (blue) using a heating rate of 1°C/min. The molar ellipticity at 222 nm was monitored. Far-UV-CD spectra of each sample from A) were recorded either immediately after the incubation at 85°C (B) or upon removal of the aggregates by centrifugation (C). Spectra were recorded at RT. Native LDH was used as control (black solid line). The dotted line represents heated LDH in the absence of polyP. At 20°C, presence of polyP had no apparent influence on the secondary structure of native LDH. (D) Percent soluble LDH after heating LDH (5.7 μM) in the absence or presence of 1 mM polyP300 to 60°C using a heating rate of 1°C/min. No significant difference in solubility was observed when shorter polyP-chains were used. The amount of soluble protein at 20°C was set to 100%. (E) Percent soluble LDH after heating LDH (5.7 μM) in the absence or presence of 1 mM polyP using different chain lengths. Samples were incubated in a thermomixer at 20°C and heated to 85°C (~10 min). The amount of soluble protein at 20°C was set to 100%.
Figure 2.
Figure 2.. PolyP forms apparently stable complexes with thermally unfolded LDH
(A) LDH (5.7 μM) was heated in the absence or presence of 1 mM polyP300 (red) to 70°C using a heating rate of 1°C/min. Samples were spun down and the soluble supernatant was analyzed by size exclusion chromatography. Samples of unheated, native LDH in the absence (black) or presence of polyP (blue) as well as polyP in the absence of LDH (green) were used as controls. Protein abundance in the eluates was determined by absorbance (solid lines) and polyP content was measured by using DAPI staining (dashed lines). LDH heated in the absence of polyP completely aggregated and was therefore not tested. (B-D) Complexes between LDH and polyP300 (LDH-polyP) were prepared as before, cooled down to RT and either analyzed directly or upon incubation with 300 nM ScPPX for 30 min at 37°C to hydrolyze polyP. (B) DAPI fluorescence measurements were conducted to determine polyP levels before and after incubation with 300 nM ScPPX. ScPPX reaction buffer (RB) was added as control, and PPX alone was tested as control as well. (C) Samples containing native LDH or pre-formed LDHpolyP complexes were either treated with nothing, RB or 300 nM ScPPX for 30 min at 37°C. After centrifugation, the insoluble pellets as well as the soluble supernatants were analyzed on SDS-PAGE and visualized by Coommassie staining. PPX alone was tested as control. (D) Far- UV-CD spectra of native LDH (black line) or LDH-polyP complexes in the absence of additives (red line), in the presence of reaction buffer (RB) (green line) or upon treatment with 300 nM ScPPX for 30 min at 37°C and subsequent centrifugation. All spectra were recorded at RT.
Figure 3.
Figure 3.. LDH-polyP complexes form defined-size oligomers
(A) Two-dimensional sedimentation analysis (2DSA) of a sedimentation velocity experiment. LDH (5.7 μM) was heated in the presence of 1 mM polyP chains from 20°C to 85°C in the thermomixer (duration: ~10 min). The samples were analyzed by analytical ultracentrifugation. Insert: Sedimentation behavior of native LDH with and without polyP14 or polyPXl. (B) Twodimension genetic analysis (GA) plots of f/f0 versus sedimentation coefficient. (C) Samples prepared for (A) were analyzed by transmission electron microscopy. An image of native LDH as well as an image of LDH-polyP130 complexes is shown as illustration (scale bar: 100 nm). (D) Representative reference-free class averages seen in both samples were selected as templates for 5 size bins (top; scale bar 10 nm). The total set of 2D class averages for each condition was subjected to reference-based matching and the total particles belonging to each bin were tabulated for both conditions (bottom).
Figure 4.
Figure 4.. Biophysical properties of LDH-polyP intermediates
LDH (5.7 μM) was heated in the absence or presence of 1 mM polyP16 (solid green) or polyP300 (solid blue) to 70°C using a heating rate of 1°C/min. Native LDH in the absence (black) or presence of 1 mM polyP16 (dashed green) or polyP300 (dashed blue) were used as control. The heated samples were spun down, and the protein concentration of the soluble supernatant was determined. (A) Fluorescence spectra of 5 μM of each sample were recorded. (B) To assess surface hydrophobicity, each sample was adjusted to a concentration of 0.5 μM LDH and supplemented with 16.5 μM bis-ANS. (C) To determine thioflavin T (ThT) binding, each sample was adjusted to a concentration of 5.7 μM LDH and supplemented with 10 μM ThT. As positive control, 5.7 μM α-synuclein fibrils incubated with 10 μM ThT was used. LDH heated in the absence of polyP completely aggregated and was not tested. (D) Two of the four subunits (cyan and pink) in rabbit muscle LDH structure (pdb: 3H3F) are shown. The regions predicted by TANGO to undergo β-aggregation are depicted in blue and red, respectively. Tryptophan residues are indicated as balls.
Figure 5.
Figure 5.. PolyP binding maintains thermally unfolded LDH refolding-competent.
(A) LDH (5.7 μM) was incubated in the presence (left panel) or absence (right panel) of 1 mM polyP130 at 60°C for 10 min. Immediately after the incubation, the samples were diluted 1:29 (0.2 μM final LDH concentration) into 10 mM KPi buffer (pH 7.5) containing 2 mM Mg-ATP, 50 mM KCl +/− 2 μM DnaK, 0.4 μM DnaJ, and 0.2 μM GrpE (KJE). Aliquots from these samples were taken at various time points, diluted 1:40 into the LDH activity assay and the initial rates of the reaction were recorded. (B) Reactivation of LDH after heating the sample from 20°C – 85°C with or without polyP130, and dilution into refolding buffer +/− KJE. (C) Relative activity of LDH heated with polyP130 at 60°C (see (A) for details) and refolded with KJE in the presence of 0, 1, 10, or 100 nM ScPPX.
Figure 6.
Figure 6.. PolyP protects UPEC strain UTI89 against high temperature treatment.
(A) UTI89 wild-type (black) and Δppk cells (red) were grown at 37°C in MOPS-glucose media. At mid-log phase (A600=0.4–0.5), cells were either left untreated (squares) or transferred into pre-warmed flasks (indicated by the arrow) at 70°C for 1 min (circles). Cells were then allowed to recover at 37°C. Growth was recorded for 8.5 h post heat-stress treatment. Insert: Survival of UTI89 wildtype and Δppk cells after 1 min heat treatment at 70°C and subsequent recovery at 37°C for 60 min. Wild-type and Δppk cells were serial-diluted and spot-titered onto LB agar plates and incubated for 15h at 37°C. (B) The accumulation of polyP after 1 min at 70°C and subsequent recovery at 37°C for 60 min was determined and normalized to the polyP accumulation in nutrient-starved cells. (C) Heat shock gene (dnaK, ibpA, ipbB) expression changes in response to brief high temperature treatment. Exponentially growing UTI89 wild-type and Δppk cells in MOPS-glucose medium (OD600 ≈ 0.4–0.5) were either left untreated or exposed to 70°C for 1 min with subsequent recovery for 15 min at 37°C. Gene expression was normalized to the expression of the housekeeping gene rrsD (encoding 16S rRNA), and fold-changes were calculated relative to the expression of each gene in the respective untreated UTI89 strain using the □□CT Experiments were performed independently at least three times.

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback