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Benchmark

Development of an in vitro assay for the detection of polymerization of the pyrin domain of ASC

Ian P Bresch‡,1,2, Dominik A Machtens‡,1,2, Thomas F Reubold1& Susanne Eschenburg*,1,2

1Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany;2Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany; *Author for correspondence: Tel.: +49 511 532 8655; eschenburg.susanne@mh-hannover.de;Authors contributed equally

BioTechniques70: 351–354 (June 2021) 10.2144/btn-2021-0011

First draft submitted: 28 January 2021; Accepted for publication: 11 May 2021; Published online: 11 June 2021

ABSTRACT

Multicomponent protein complexes called inflammasomes play a major role in the innate immune system by activating proinflammatory cy- tokines and promoting a highly inflammatory form of programmed cell death, called pyroptosis. A hallmark of the function of the nucleotide- binding domain, leucine-rich repeat and NLRP3-mediated inflammasome assembly is the polymerization of ASC into large filaments. The ASC filaments recruit and activate procaspase-1 by induced proximity. We developed anin vitroassay for monitoring the polymerization of the pyrin domain of ASC by microscale thermophoresis. We have validated the assay by analyzing the effects of buffer conditions, mutations of ASC and the use of seeds on the polymerization behavior of ASC.

METHOD SUMMARY

We developed a new assay for the detection of the polymerization of ASCPYDin vitro. Using microscale thermophoresis, we monitored the thermophoretic movement of Atto 488-labeled ASCPYD. The assay allows a fast and sample-efficient quantification of the formation of ASCPYD filaments.

KEYWORDS:

ASC•immune response•inflammasome•innate immunity•microscale thermophoresis•polymerization

The innate immune system must perform a rapid signal amplification after the detection of minimal amounts of pathogen- or danger- associated molecular patterns. For this task, the innate immune system employs germline-encoded pattern recognition receptors (PRRs). Signaling cascades within the inflammatory and apoptotic machinery use the simple death domain fold that mediates homo- typic interactions[1]. The adaptor protein ASC consists of two death domains, the pyrin domain (PYD) and the caspase activation and recruitment domain (CARD), which are connected by a flexible linker[2]. Activated PRRs like NLRP3 or AIM2 assemble into multimeric inflammasomes that recruit ASC via PYDs and induce the polymerization of ASC into filaments[3]. Subsequently, the flexible CARDs connect the PYD filaments and form a stable speck-like complex that functions as a supramolecular organizing center for caspase-1 processing[4]. Processed caspase-1 then activates pro-IL-1␤and pro-IL-18 to amplify the proinflammatory cascade[5]. Additionally, ac- tivation of pore-forming gasdermin D may lead to pyroptotic cell death[6]. The AIM2 inflammasome is involved in detection of viruses via recognition of cytosolic dsDNA, whereas the NLRP3 inflammasome plays a role in the recognition of various pathogens as well as in sterile inflammation[7].

The formation of ASC specks was shown first by overexpression in eukaryotic cells[8]and was later confirmed in cell-free sys- tems[3,4]. The NMR structure of ASC and the cryo-electron microscopy structures of ASCPYDfilaments[3,9,10], together with site-directed mutagenesis experiments[11]and immunofluorescence microscopy ofin vivospecks[12], support the hypothesis that ASCPYDbuilds the backbone of the filaments and the CARD of ASC crosslinks the filaments and recruits procapase-1. Single-molecule fluorescence[13] and anin vitroactivity assay[14]showed the prion-like behavior of ASC specks leading to an ‘all or nothing’ response.In vitroexperi- ments revealed that ASC is able to polymerize without activators depending on its concentration and that this starting concentration is decreased by active PRRs or ASC seeds[13]. A high-throughput assay based on fluorescence anisotropy has been used to screen for inhibitors of the formation of ASCPYDfilaments and its pH dependence[15].

Here we developed anin vitroassay for the detection of the polymerization of ASCPYDusing microscale thermophoresis (MST). MST detects movement along a temperature gradient of fluorescently labeled molecules. The thermophoretic movement depends on the size, charge and hydration shell of the molecules. After complex formation, one or more of these parameters are changed, leading to differences in the thermophoretic behavior, which offers the possibility to determine dissociation constants. This makes MST a highly sensitive method for analyzing the binding of two or more molecules[16]. Furthermore, MST has been used previously to monitor the poly- merization of actin into filaments in real time, which hints at a general applicability of the method for the investigation of polymerization processes[17].

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Benchmark

Table 1. Determined concentrations at which ASCPYDstarts to polymerize.

Protein Deviation of standard

conditions

cpolymerization(µM) Protein Deviation of standard

conditions

cpolymerization(µM)

ASCPYD-WT 1.37±0.02 ASCPYD-L25A 7.44±0.12

ASCPYD-WT 50 mM KCl 1.27±0.01 ASCPYD-E13R-K21E-K22E 31.80±1.70

ASCPYD-WT 300 mM KCl 3.09±0.06 ASCPYD-WT 25 mM HEPES (pH 8.0),

50 mM NaCl

1.34±0.07

ASCPYD-WT 600 mM KCl 8.68±0.10 ASCPYD-WT 25 mM HEPES (pH 8.0),

50 mM NaCl+ASC seeds

0.64±0.04

ASCPYD-WT pH 5.0 1.90±0.05 GAMGGSEF-ASCPYD 12.08±1.27

Concentrations were determined using the Hill1 fit with the Levenberg–Marquardt iteration algorithm. Errors represent standard error of the determined concentration.

WT: Wild-type.

The software NT Control (v. 2.2.1) was used to control the MST device (Monolith NT.115Pico, NanoTemper Technologies, Munich, Germany). The device contains light-emitting diodes, an IR laser and a fluorescence detector (Figure 1A). The IR laser produces a tem- perature gradient within the capillary of 2–6C, inducing the thermophoretic movement[16]. For this assay, recombinantly expressed and purified MBP-ASC(1-105)-Cys (later referred to as MBP-ASCPYD) was used. This construct contains the PYD and the linker between PYD and CARD and has already been utilized in earlier experiments[3]. Labeling of the C-terminal cysteine of MBP-ASCPYDwas performed with a 2.5-fold molar excess of ATTO 488–maleimide in a buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl and 1 mM TCEP for 1 h in the dark at room temperature. Unbound dye was removed by dialysis against a buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl and 2 mM DTT.

The reaction volume for each capillary was 20µl, and the standard buffer for the MST measurement contains 50 mM Tris/HCl (pH 8.0), 150 mM KCl, 5 mM DTT, 25 nM MBP-ASC(1-105)-ATTO 488 and 0.05 mg/ml TEV protease. After mixing the buffer with different concentrations of unlabeled MBP-ASCPYD, the reaction solutions were loaded into the capillaries. The capillaries with the samples were incubated for 1 h at 22C, allowing the TEV protease to cleave off the polymerization preventing MBP tagging. Afterward, the samples were incubated for an additional 30 min at 37C to complete the polymerization. We confirmed that an incubation time of 30 min is sufficient and validated our assay for conditions in the pH range 5.0–9.0 including a KCl concentration of up to 600 mM (Supplementary Figure 2). The IR laser power was set to 60% and the blue light-emitting diode to 70% power. To exemplify the raw data, the fluorescence traces of the polymerization experiments with different KCl concentrations are shown in Figure 1B. The intervals within the lines of equal color were selected for the determination of F1(red lines) and F0(blue lines), respectively. F1and F0were used to calculate the normal- ized fluorescence, which was plotted against the concentration of unlabeled ASCPYD. The Hill fit for calculation of the concentration at which ASCPYDstarts to polymerize was used in earlier experiments to describe the supramolecular self-assembly[18]. The determined concentrations are listed in Table 1. The respective experiments were performed four times each.

The decrease of the intracellular potassium concentration has been described as a prerequisite for the activation of the NLRP3 inflammasome[19]. We have shown that increased KCl concentrations decrease the ability of ASCPYDto polymerize (Figure 1C). To exclude that this result was influenced by inefficient TEV cleavage at high salt concentrations such that less ASCPYDwas available for polymerization, we confirmed the complete cleavage of MBP-ASCPYDat 600 mM KCl (Supplementary Figure 1B). Thus the observed decrease in polymerization efficiency can be solely attributed to a direct effect of the increased KCl concentration.

To ensure that the decrease in the normalized fluorescence was caused by polymerization and not by aggregation, we cloned and purified two different mutants of ASCPYD. ASCPYD-L25A only polymerizes at higher concentrations and was used in earlier NMR experi- ments[1]. ASCPYD-E13R-K21E-K22E is a combination of two described ASCPYDmutants impairing two of the three interaction interfaces of the PYD–PYD interaction[3]. ASCPYD-L25A requires a fivefold higher concentration to start its polymerization, while ASCPYD-E13R-K21E- K22E requires a 20-fold higher starting concentration than wild-type ASCPYD(Figure 1D). The mutations do not influence the structure of ASCPYD[1,3]. The gradual decrease in the normalized fluorescence suggests that the impairment of two of the three interaction in- terfaces prevents an ‘all-or-nothing’ behavior leading to more intermediate states between a monomer and a filament. Together, these results show that the effect is not due to aggregation but due to polymerization of ASCPYD.

The effect of changing the pH value of the buffer is shown in Figure 1E. A lower pH value increases the concentration at which ASCPYD starts to polymerize, from 1.4 to 1.9µM, which is in agreement with the increased solubility at a pH of 3.8, as described earlier[9]. ASC polymerization cannot be detected at pH values lower than 5.0, because the TEV protease is inactive at low pH values[20]. We confirmed the completeness of the TEV cleavage at different pH values of 5.0–9.0 (Supplementary Figure 1), which suggests that the assay can be used over a wide pH range and that a digestion time of 60 min followed by an incubation time of 30 min is sufficient under our conditions. Generally, the extent of polymerization of ASCPYDis a function of various parameters, including ASCPYDconcentration, TEV concentration, KCl concentration, pH and incubation time. Although we have shown that the assay is suited to assessing the influence of different parameters on ASCPYDpolymerization, we advise potential users to carefully adjust and verify the experimental conditions for more specialized settings. In particular, the activity of individual noncommercial TEV preparations should be tested.

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0.86 0.88 0.90 0.92 0.94 0.96 0.98 1.00 1.02

0.0 0.2 0.4 0.6 0.8 1.0

FInorm

1.2

ASC concentration [µM]

0.1 1

ASC concentration [µM]

1 10 100

Excitation light

Dichroic mirror IR laser

Objective

Capillaries

0.0 0.2 0.4 0.6 0.8 1.0

FInorm

1.2

ASC (1–105)-WT ASC (1–105)-L25A

ASC (1–105)-E13R-K21E-K22E

0 10 20 30

Time [s]

50 mM KCI 150 mM KCI 300 mM KCI 600 mM KCI

ASC concentration [µM]

1 10 100

50 mM KCI 150 mM KCI 300 mM KCI 600 mM KCI

0.0 0.2 0.4 0.6 0.8 1.0

FInorm

1.2

ASC(1-105)

ASC(1-105)+ASC seeds

ASC concentration [µM]

1 10

0.0 0.2 0.4 0.6 0.8 1.0

FInorm

1.2

pH 8.0 pH 5.0

ASC concentration [µM]

1 10 100

0.0 0.2 0.4 0.6 0.8 1.0

FInorm

1.2

-0.2

ASC(1-105)

GAMGGSEF-ASC (1-105) FInorm

Figure 1. Detection of the polymerization of ASCPYDby microscale thermophoresis. (A)Schematic representation of the MST device.(B)Raw fluorescence traces of ASCPYDpolymerization in different KCl concentrations. The areas within the lines were used for the determination of the fluorescence before (blue) and after (red) thermophoresis.(C)ASCPYDpolymerization in solutions containing different concentrations of KCl. Effects of (D)structure-based mutations,(E)pH of the buffer and(F)the addition of polymerized ASC to the samples, and of(G)additional amino acids (GAMGGSEF) in front of the N-terminus, on ASCPYDpolymerization. Each experiment was performed four times. Origin 2019 (OriginLab, MA, USA) was used to prepare all graphs.

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Benchmark

The ability of polymerized ASC or ASC seeds to activate monomeric ASC is an important mechanism for the spreading of the in- flammatory reaction to neighboring cells[13]. We showed that the addition of ASCPYDseeds to the samples halved the concentration at which ASCPYDstarted to polymerize (Figure 1F). For these measurements, a buffer composed of 25 mM HEPES (pH 8.0), 50 mM NaCl and 5 mM DTT was used. This buffer has been used earlier for experiments showing the effect of ASC seeds[13]. To generate ASCPYD seeds, 20µM ASCPYDwere incubated with 2µM TEV protease and then added to the samples for the MST measurements with a 1:200 dilution.

Moreover, we found that the amino acid composition of the linker sequence preceding the N-terminus could impair the polymerization of ASCPYD(Figure 1G). Using a construct that possesses a GAMGGSEF sequence at the N-terminus after TEV digestion, we observed a drastically decreased ability to polymerize. This construct starts to polymerize at 12µM, which is approximately ten-times higher compared with a construct with a short N-terminal GH motif, after TEV digestion. We hypothesize that the positively charged surface at the N-terminus formed by the critical residues Arg3 and Arg5 is blocked by glutamate of the linker peptide[3]. This would impair the most important interaction interface and so decrease the ability of ASC to polymerize.

We have developed and validated anin vitroassay for observing the polymerization of ASCPYDbased on MST. Our assay allows the determination of apparent binding constants of the assembly reaction. Moreover, we have shown that the influence of ASC mutations as well as of different parameters including pH and salt concentration can be assessed. Thus our assay may be suited to characterize small molecule effectors that decrease ASCPYDpolymerization for the development of potential anti-inflammatory agents.

Suplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.

2144/btn-2021-0011

Author contributions

I Bresch performed experiments and analyzed the data. D Machtens conceived and designed the experiments and analyzed the data. I Bresch, D Machtens, T Reubold and S Eschenburg interpreted the data and wrote the manuscript. S Eschenburg supervised the study.

Financial & competing interests disclosure

The project was financially supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2155 ‘RESIST’ – Project ID 39087428. I Bresch is a graduate student in the MD/PhD program Molecular Medicine of the Hannover Biomedical Research School (HBRS). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Open access

This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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