An innate interaction between IL-18 and the propeptide that inactivates its precursor form
An innate interaction between IL-18 and the propeptide that inactivates its precursor form
Published: xx xx xxxx Uncontrolled secretion of mature interleukin (IL)-1? and IL-18 is responsible for severe autoinflammatory or autoimmune disorders and various allergic diseases. Here we report an intramolecular interaction between IL-18 and its propeptide, which is proteolytically removed from its precursor proIL-18 during maturation. The intramolecular interaction was recapitulated intermolecularly using recombinant propeptide. These results suggest the possibility of developing a novel class of peptide-based IL-18 inhibitors that could serve as therapeutic agents for IL-18-related inflammatory diseases.
Interleukin (IL)-18 is a proinflammatory cytokine belonging to the IL-1 family (IL-1F) and potently stimulates
interferon (IFN)-? production to protect hosts against infections1,2. All IL-1F ligand agonists, including IL-18, are
synthesized in precursor forms (proIL-1F)3 bearing N-terminal propeptide sequences (PPs) with lengths varying
from 4 to 116 amino acids. The most of proIL-1Fs are either completely inactive or less active, and the
maturation step, in which PP is removed via enzymatic cleavage, is necessary for full activation. For instance,
inflammatory IL-18 and IL-1? are produced as precursors called proIL-18 and proIL-1?, respectively. The precursors
remain inactive in the cytosol until a signal, inflammasome activation, induces maturation. The inflammasome is
a multi-protein assembly composed of three proteins, a nucleotide-binding oligomerization domain (NOD)-like
receptor, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1, and is formed upon
detection of pathogens or other harmful substances, such as reactive oxygen species and urate crystals, in the
cytosol. The assembly then autocatalyzes activation of the cysteine protease caspase-14, which removes PPs from
the precursors to produce IL-1? and IL-18. These mature forms are then secreted extracellularly to exert their
biological functions; however, the mechanism underlying this secretion remains controversial5?7. Secreted IL-1?
and IL-18 bind to their receptor pairs (IL-1RI/IL-1RAcP8 and IL-18R?/IL-18R?9, respectively) on target cells to
initiate intracellular MyD88-dependent signaling. This ultimately activates NF-?B, which upregulates expression
of various inflammatory cytokines. In a sense, IL-1? and IL-18 PPs securely lock in the cytokines? powerful
proinflammatory activity so that they cannot readily trigger severe inflammatory diseases10?13. In fact, proIL-1? and
proIL-18 are exposed to the extracellular space upon pyroptotic cell death; however, maturation is still required
for them to engage their receptors, eliciting intracellular signaling and the inflammatory response14?16. Especially
in the case of IL-18, the fast process of enzymatic maturation of the stored precursor, rather than the slower
production of mature proteins by transcription and translation, may facilitate rapid release of active cytokine in
response to invading pathogens. In contrast, other IL-1F members, such as proIL-1? and proIL-33, are somewhat
active even when their PPs remain intact. They can activate their receptors on target cells when released from
dying cells although the activity of proIL-33 is weaker than that of its mature form14,17.
Recent studies have shown that inflammasomes play a crucial role in regulating IL-1? and IL-18 secretion, and
many genetic disorders in their components are responsible for high circulating levels of these cytokines,
resulting in autoinflammatory syndromes. The most typical case is cryopyrin-associated periodic syndrome (CAPS),
in which specific mutations in the NOD-like receptor NACHT, LRR, and PYD domains-containing protein 3
(NLRP3)10 induce uncontrolled NLRP3 inflammasome activation, which eventually leads to chronic
inflammation because of IL-1? hyper-production. Similarly, certain mutations in the NLR family CARD domain-containing
protein 4 (NLRC4) evoke periodic fevers of lethal macrophage-activating syndrome (MAS)11?13,
in which patients suffer from extremely high blood levels of IL-18 in addition to elevated levels of IL-1?.
Interestingly, IL-18 levels in NLRC4-MAS patients remain high for a long period, even after IL-1? blockade.
These findings reinforce the concept that PPs as important regulators of proinflammatory IL-1? and IL-18.
In addition to NLRC4-MAS, IL-18 is associated with various severe chronic inflammatory diseases such as
CAPS, familial Mediterranean fever, adult-onset Still?s disease (AOSD), pyogenic arthritis, pyoderma
gangrenosum, acne syndrome, and systemic juvenile idiopathic arthritis with MAS18?22. High blood concentrations of
IL-18 are also found in patients with allergic diseases, including bronchial asthma, atopic dermatitis,
inflammatory bowel disease, and the X-linked inhibitor of apoptosis (XIAP) deficiency23?26. Genetic polymorphisms have
also been reported in the IL-18 or IL-18 receptor genes of patients with autoimmune diseases, allergic reactions,
and neurological/metabolic syndromes27?31.
Neutralizing IL-1? by using either anti-IL-1? antibodies (Canakinumab) or engineered soluble
receptors (Rilonacept), or by antagonizing the receptor with IL-1Ra (Anakinra), treat IL-1?-related
autoinflammatory diseases effectively32?35. Similarly, anti-IL-18 antibodies inhibit the development of atopic dermatitis and
asthma-like phenotypes in mouse models36,37. A recombinant human IL-18-binding protein (IL-18BP, Tadekinig
Alfa), an endogenous antagonist of IL-18, is also in mid-late phase clinical trials for AOSD, XIAP deficiency and
Here, we demonstrate an intramolecular interaction between the mature region of IL-18 and its PP, providing
a solid framework for further investigations of PPs? roles in IL-1F ligands? molecular function. These results also
suggest the possibility of developing a new class of peptide-based drugs to treat IL-1F-associated inflammatory
Construction of expression vectors. The coding region of the full-length human IL-18 precursor
(proIL18, NM_001562.3, residues 1?193) was amplified from a human cDNA library by a polymerase chain
reaction and cloned into a pET-28a vector (Novagen, WI, USA), which was engineered to contain a 6? His-tagged
N-terminal small ubiquitin-like modifier (SUMO)-3 (NM_006936.2, residues 14?92) for protein expression and
purification. The plasmids for mature IL-18 (residues 37?193) and proIL-18PP (residues 1?36) were derived from
the proIL-18 construct by deleting residues 1?36, or by replacing Y37 with a stop codon, respectively, using a
KOD -Plus- Mutagenesis kit (Toyobo, Osaka, Japan). The constructs for the N-terminal-truncated proIL-18
variants (proIL-18?Ns), namely, proIL-18?8N (residues 9?193), proIL-18?10N (residues 11?193), proIL-18?12N
(residues 13?193), proIL-18?13N (residues 14?193) and proIL-18?22N (residues 23?193), were prepared in the
same manner as the IL-18 expression plasmid.
Protein expression and purification. The expression vectors for proIL-18, proIL-18?Ns, IL-18 and
proIL-18PP were transformed into E. coli BL21(DE3) cells, and the bacteria were grown in LB?medium or M9
minimal media containing either?15NH4Cl/12C6H12O6 or 15NH4Cl/13C6H12O6 at 37 ?C in the presence of 25 ?g/
mL kanamycin. Cultures were cooled on ice to 18 ?C when the optical density at 600 nm reached 1.0, and
protein expression was induced by adding 1.0 mM IPTG. Cells were harvested after incubating for 16 h at 18 ?C,
resuspended at 4 ?C in lysis buffer (20 mM Tris.HCl, pH 8.0, 150 mM NaCl, 20 mM imidazole and 0.5?2 mM
DTT), and supplemented with 1 mM PMSF for proIL-18, proIL-18?Ns and IL-18 or with cOmplete EDTA-free
Protease Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany) for proIL-18PP. After sonication on ice
and centrifugation, the supernatant was applied to an Ni affinity column (cOmplete His-Tag Purification Resin,
Roche Applied Science) equilibrated with lysis buffer. After washing with lysis buffer, followed by detergent buffer
(20 mM Tris.HCl, pH 8.0, 150 mM NaCl, 20 mM imidazole, 1% Triton X-100 and 2 mM DTT), high-salt buffer
(20 mM Tris.HCl, pH 8.0, 1 M NaCl, 20 mM imidazole and 2 mM DTT) and then again with lysis buffer, the
His-SUMO tag was removed by digestion with a homemade SUMO-specific protease GST-SENP2 for 16h at 4 ?C
on column. The protein solution was eluted and allowed to flow through a glutathione sepharose (GE Healthcare,
Little Chalfont, UK) to remove the protease. The flow through was further purified using a HiTrap Q anion
exchange column (GE Healthcare) and eluted with an NaCl gradient from 50 mM to 500 mM. This was followed
by a size-exclusion chromatography on a HiLoad 16/60 Superdex 75 column (GE Healthcare). The gel-filtration
column was equilibrated with either NMR buffer (20 mM potassium phosphate, pH 6.0, 150 mM KCl and 1 mM
TCEP) for proIL-18, proIL-18?Ns and IL-18 or with 300 mM ammonium acetate for proIL-18PP. The
proIL18PP eluate was quantitated, aliquoted and lyophilized.
NMR spectroscopy. NMR spectra were measured on Bruker Avance II 700 MHz, Avance 600 MHz and
Avance III 500 MHz spectrometers equipped with cryogenic probes. The samples for NMR analyses were
dissolved in either NMR buffer or caspase-1 reaction buffer (20 mM HEPES-Na pH 7.4, 100 mM NaCl, 1 mM
EDTA and 10 mM DTT), which was exchanged by dialysis, and 5 v/v % D2O was added before measurements.
To detect interactions between IL-18 and proIL-18PP, 28.4 nmol of lyophilized [15N]-proIL-18PP was directly
dissolved in 300 ?L of either NMR buffer or 320 ?M non-labeled IL-18 in NMR buffer. Reverse detection was
performed with 240 ?L of 50 ?M [15N]-IL-18 in the presence and absence of approximately 5 molar equivalents
(eq.) of proIL-18PP in caspase-1 reaction buffer. Chemical shift assignments for free proIL-18PP were based on
a HNCACB/CBCA(CO)NH data set. To record 1H-15N SOFAST HMQC spectra with in situ caspase-1 cleavage,
10 units of caspase-1 (Enzo Life Sciences, NY, USA) was added to 300 ?L of 200 ?M proIL-18. Each 2D spectrum
was collected for approximately 71 min, and 20 spectra were recorded over approximately 24 hours after adding
caspase-1. For comparison, a reaction mixture was prepared, using the same lot of proteins and under the same
conditions as the in situ NMR experiment, and was analyzed by SDS-PAGE at various time points to confirm the
enzymatic activities. To compare the thermotolerance of IL-18 and proIL-18, 1H-15N SOFAST HMQC spectra
were sequentially recorded at 40 ?C, 45 ?C and 50 ?C. The spectra were processed using NMRPipe41 or Bruker
TopSpin version 3.5pl7 and analyzed using Sparky42 and CcpNmr Analysis43 version 2.4.2.
Circular dichroism (CD) spectroscopy. CD spectra were collected on a J-700 spectrophotometer (JASCO,
MD, USA) from 250 to 200 nm at 0.1 nm intervals. The proIL-18 and IL-18 concentrations were adjusted to
0.2 mg/mL in NMR buffer and data were reported as molar ellipticity ? (deg cm2/dmol). For thermal studies, the
molar ellipticity at 215 nm were recorded from 20 ?C to 80 ?C at a heating rate of 1 ?C/min, with 0.2 ?C intervals.
Data were processed using Igor Pro 126.96.36.199 (WaveMetrics, OR, USA) using moving averages of 1 nm and 1 ?C for
the comparison of spectral pattern and thermal scanning, respectively.
Detection of the interaction between IL-18 and proIL-18PP. We first prepared mature IL-18
and proIL-18, the precursor of IL-18, for analysis by NMR spectroscopy. All recombinant IL-18 variants were
expressed with an N-terminal SUMO tag to precisely produce the polypeptide sequences of interest; an
analogous method has been reported to produce fully bioactive IL-1844. The 2D 1H-15N HMQC spectrum of
[15N]IL-18 prepared in this way was essentially identical to the one we obtained in our previous structural studies
of IL-189,45, indicating that the sample protein had the proper tertiary structure to form the signaling ternary
complex. ProIL-18 exhibited a monomeric, mono-disperse nature upon size-exclusion chromatography, as did
IL-18 (Supplementary Fig.?1a and ref.46). However, the 2D 1H-15N HMQC spectrum of [15N]-proIL-18 was
dramatically different from [15N]-IL-18 (Fig.?1a and ref.9), suggesting that the PP (Fig.?1b right, residues 1?36) makes
extensive contact with the mature region (Fig.?1b right, residues 37?193) and/or prevents the proper folding of
the mature region via certain intramolecular interactions. The cross-peaks were substantially broadened, likely
reflecting relatively weak interactions and low structural homogeneity of the molecule. In terms of function, the
distinct structural state of proIL-18, revealed by the NMR spectra, is consistent with its inability to activate IL-18
receptors, triggering IL-18 signaling47,48. Since the receptors recognize large surface areas of IL-189, the clearly
distinct structure of proIL-18 should hamper binding.
To determine whether the observed intramolecular interaction can be reproduced intermolecularly, we next
examined the binding between a peptide comprising the PP sequence (proIL-18PP, Supplementary Fig.?1b) and
mature IL-18. The structure of recombinant [15N]-proIL-18PP was completely disordered since the chemical shift
dispersion of the main-chain amide protons in the 2D 1H-15N HMQC spectrum was narrow and lay in a range
of 8.4 to 8.0 ppm49 (Fig.?1c and Supplementary Fig.?2a). The secondary structure propensity was evaluated based
on the backbone heavy atom assignments of free [13C/15N]-proIL-18PP (Supplementary Fig.?2b), confirming that
there is essentially no secondary structure in the free peptide.
However, the NMR spectrum of [15N]-proIL-18PP changed substantially in the presence of three molar
equivalents of IL-18, with new cross-peaks appearing at a ?(1H) of 9.5?8.5 ppm, indicating specific interaction between
proIL-18PP and IL-18 and the formation of an ordered structure in the peptide (Fig.?1c). These new peaks can
be superimposed on certain peaks of [15N]-proIL-18 (Fig.?2a, top left), suggesting that the peptide experiences
the same environment as proIL-18. In other words, proIL-18PP?s binding to IL-18, at least partly, recapitulates
the native intramolecular interactions within proIL-18. Although we were unable to assign the new cross-peaks,
a certain portion of proIL-18PP was thought to form a ?-strand structure based on the amide 1H chemical shifts
of the peaks (Supplementary Fig.?2c). This interpretation agrees with secondary structure prediction, where
Cys10-Ile19 and Thr22-Ala27 adopt short ?-strand configurations (Supplementary Fig.?2d). Intriguingly,
1H15N NMR signals from amino acids residues in the predicted ?-strands were substantially broadened during the
Based on the 1H-15N cross-peak intensities of [15N]-proIL-18PP in the presence of three molar equivalents of
IL-18, the dissociation constant (KD) of the interaction was evaluated as >36 ?M. This affinity is in the moderate
range, however, in full-length proIL-18, these two regions are connected into a single molecule by a peptide bond,
so that they should assemble more tightly. As shown in Fig.?1d, when three molar equivalents of IL-18 were
present, the cross-peak intensities of?free [15N]-proIL-18PP decreased, which was substantial for amino acid residues
from Asn9 to Ala27. This observation suggests this region that mainly interacts with IL-18, while the remainder
of the peptide does not.
To further explore the region in the PP sequence that is essential for IL-18 binding, we next recorded the
1H-15N HMQC spectra of a series of N-terminally truncated proIL-18 derivatives (proIL-18?Ns) and compared
them to full-length proIL-18 and mature IL-18 (Fig.?2). [15N]-proIL-18?8N, in which eight N-terminal residues
were truncated, produced a very similar spectral pattern to [15N]-proIL-18 (Fig.?2 middle) with the signature
cross-peak appeared during [15N]-proIL-18PP/IL-18 titration (squared peak in Fig.?2). This observation indicates
that the first eight residues are dispensable for the interaction between the pro and mature regions of proIL-18.
Because the spectral comparison did not reveal any obvious differences in the locations of the well-isolated
cross-peaks, the first eight amino acid residues of proIL-18 may adopt a random coil whose cross-peaks are
presumably buried in the crowded and indistinguishable region at approximately a ?(1H) of 8.4?8.0 ppm. Additional
derivatives were also tested; intriguingly, after deleting the first ten residues of proIL-18 (proIL-18?10N, Fig.?2
right), the spectra was broadened substantially, presumably because of intermediate states between proIL-18 and
IL-18. Furthermore, the reference cross-peak of full-length proIL-18 began to change (squared peak in Fig.?2).
In addition, deletion of the first 12 residues (proIL-18?12N, Fig.?2 bottom left) completely altered the spectrum
to resemble the mature protein, indicating a critical importance of residues near Ile11. Further deletion did not
produce major spectral changes, and proIL-18?13N and proIL-18?22N yielded essentially the same spectra as
proIL-18?12N (Supplementary Fig.?3). Taken together, these data suggest that residues 9?11 play a central role
in the interaction, although residues 12?27 probably also participate in IL-18 binding.
In cells, proIL-18 maturation occurs via cleavage by caspase-1. To obtain insights into this reaction, we
performed in situ NMR analysis of the maturation reaction in a test tube, wherein 1H-15N HMQC spectra of
[15N]-proIL-18 were sequentially recorded in the presence of active caspase-1 (Fig.?3a?c and Supplementary
Fig.?4a). The majority of [15N]-proIL-18 was digested within one hour and the reaction was complete after
24 hours, according to SDS-PAGE analysis (Fig.?3a). However, even in the NMR spectrum recorded 24 hours after
the addition of caspase-1, essentially all cross-peaks for [15N]-proIL-18 remained, although their intensity had
decreased by approximately 50% (Fig.?3b,c). In addition, new cross-peaks corresponding to mature IL-18 were
apparent, indicating that some fraction of proIL-18 was structurally converted to the mature form (Fig.?3d). These
observations indicate that ~50% of the generated proIL-18PP was still in complexed with the mature region, even
after cleavage by caspase-1. In fact, considering the initial concentration of proIL-18 in this experiment (200 ?M),
the KD can be estimated from the NMR spectra (Fig.?3b) as approximately 100 ?M, similar to the value determined
by titration (Fig.?1c). To confirm the spectral pattern of the mature region of IL-18 in the peptide-bound state,
we measured 1H-15N HMQC spectrum of [15N]-IL-18 in the presence of approximately five molar equivalents of
non-label proIL-18PP. The yielded spectrum had the similar features to proIL-18, as was in the caspse-1 cleaved
experiment but with no signal from free and complexed proIL-18PP (Fig.?3e and Supplementary Fig.?4b).
ProIL-18 is more stable than mature IL-18. To compare the secondary structures and folding properties
of proIL-18 and IL-18, we collected circular dichroism (CD) spectra in NMR buffer containing 150 mM NaCl
(Fig.?4). Consistent with the reported ?-trefoil structure9,45 with abundant ?-hairpins and short connecting loops,
IL-18 produced a broad, negative peak at 208 nm (Fig.?4a, red) with a spectral pattern very similar to the reported
CD spectra of IL-1Ra and IL-36?50,51. The negative band of proIL-18 was broadened and shifted to a lower
wavelength relative to IL-18 (Fig.?4a, blue), indicating a slightly higher random coil content in proIL-18. We then
performed thermal scanning by recording CD values at 215 nm50 from 20 ?C to 80 ?C at a heating rate of 1 ?C/
min (Fig.?4b). IL-18 denatured at a melting temperature (Tm) of 55 ?C, while the CD value of proIL-18 remained
unchanged at this point. The CD value of proIL-18 began to drop near 58?C did so gradually to approximately
68 ?C, where it became stable. This decrease in the CD value can be attributed to soluble aggregation52 and the
midpoint of the transition between the two states was 63 ?C.
The same trend was observed in the high-temperature NMR experiments (Fig.?5a,b). IL-18 formed an
irreversible white precipitate at 50 ?C after the course of 73 min of measurement, and all cross-peaks disappeared
from the spectrum. In contrast, the spectral pattern of proIL-18 was essentially the same between 40 ?C and
50 ?C, and the signal intensity only decreased by 30% (Fig.?5c). The Tm was higher than 50 ?C for IL-18 in the
temperature scan with the CD measurement (Fig.?4b), whereas the protein precipitated at 50 ?C in the NMR study.
The discrepancy between these two experiments could be attributable to the difference in the experimental time
scales; the temperature in the CD measurements was too fast to allow establishment of a thermal equilibrium state
at each temperature. Nevertheless, in both cases, proIL-18 exhibited higher thermostability than IL-18, marking
a sharp contrast to proIL-1?, which is substantially less stable than its mature form53.
Previous structural studies have revealed that mature IL-18?s N-terminal tyrosine residue is relatively proximal,
in the binary complex, to the glycan chain on Asn297 of the primary receptor IL-18R?9,54. Hence, it was likely
that the 36-amino acid PP of proIL-18 would sterically hinder binding and so the formation of the signaling
complex. In this study, we provided a more detailed structural view of proIL-18 by demonstrating that the PP
region extensively interacts with the mature region of proIL-18. Chemical shifts of amide 1H suggest that at least a
part of the PP region likely adopts a ?-strand configuration in proIL-18, implying ?-sheet formation between the
PP and mature regions. Nevertheless, the resemblance of the CD spectra indicated similar secondary structure
content between proIL-18 and IL-18 except for random coils. Thus, a part of the mature region?s ?-trefoil structure
might have been distorted in proIL-18. The extensively interacting PP region may add additional complexity to
determining IL-18?s bioactivities. Due to its potent proinflammatory activity, the IL-18 activity must be tightly
regulated. In fact, under normal conditions, blood IL-18 is bound to natural inhibitors, the IL-18 binding
protein (IL-18BP)30 and soluble IL-18Rs (sIL-18R? and sIL-18R?)55, which neutralize its activity. However, in some
detrimental autoimmunity or autoinflammatory conditions18?26, systemic IL-18 levels are elevated and far exceed
those of their antagonists, resulting in the appearance of the active cytokine. Similar tight regulation has also been
observed in the IL-1? system, where soluble IL-1 receptors (sIL-1RI and sIL-1RII), a decoy cell-surface
receptor (IL-1RII) and an IL-1 receptor antagonist (IL-1Ra) play anti-inflammatory roles in vivo. Previous structural
characterization of proIL-1? suggested that its PP region prevents the completion of ?-trefoil barrel folding by
directly interfering with the mature region, although the PP largely adopts a random coil conformation53, and
thus binding of immature IL-1? to its specific receptors is abrogated. Therefore, PP regions might share a
common mechanism for securely suppressing IL-1?/IL-18?s proinflammatory activities.
An intriguing difference between proIL-18 and proIL-1? concerns their folding stability. Our CD and NMR
studies have revealed that proIL-18 has an enhanced thermostability relative to the mature protein, which may be
advantageous in preserving the precursor in the cytosol for longer durations. This is in sharp contrast to proIL-1?,
whose PP in the immature form has been proposed to prevent full barrel formation of the mature region,
destabilizing the protein53. This difference distinguishes two closely related members of IL-1F, in which IL-18 mRNA
and protein are constitutively expressed and stored in the cytosol, whereas IL-1? is transcribed and produced
on-demand by various stimuli, such as lipopolysaccharides, and processed to the mature form immediately56.
IL-1??s instability may be related to its secretion pathway, where an unconventional autophagy-mediated
mechanism has been proposed to be responsible for IL-1? release. In this pathway, less stable proIL-1? is more
susceptible to protease-dependent degradation and is, thus, prevented from being secreted6. ProIL-18?s higher stability
implies that a similar pathway is not used for IL-18 secretion.
The present NMR study demonstrates that proIL-18PP binds to IL-18 to produce dramatic spectral changes in
IL-18 (Figs?1?3) that are comparably extensive to those observed when [15N]-IL-18 binds the extracellular region
of IL-18R?9. Because IL-18BP, a potent IL-18 inhibitor, only mimics one of the three subdomains of IL-18R?
when binding IL-18, it is possible that proIL-18PP interferes with the binding between IL-18 and IL-18R?.
However, the intermolecular binding between proIL-18PP and IL-18 has a lower affinity (tens to 100?M) than
the intramolecular binding between the pro and mature region of proIL-18 because there was no peptide bond
between them. Thus, their interaction should be short-lived. Furthermore, peptides are generally susceptible to
protease digestion in cell culture and in vivo. Hence, proIL-18PP?s inhibitory effect on IL-18 activities may not be
very strong. Nevertheless, optimizing its binding properties by changing its amino acid sequence or reformatting
it into peptibodies57 containing multiple proIL-18PP-like sequences may produce more potent inhibitory
compounds. Because proIL-18PP is an endogenous polypeptide generated from proIL-18 upon maturation, its
immunogenicity and toxicity should be low if such modifications were minimal; this would be highly beneficial for
clinical application, and so proIL-18PP may represent an excellent seed compound to develop IL-18-neutralizing
drugs in the future. In this context, our extensive NMR analysis has provided valuable information, i.e., that the
central three amino acid residues in proIL-18PP play a key role in mediating its interaction with mature IL-18.
IL-18?s natural inhibitory mechanism could be applied to other IL-1F ligands, which will affect our
understanding of IL-1F biology and the development of therapeutics for IL-1F-related diseases.
All the unique materials and relevant raw data in this study are available from the corresponding authors upon
The authors thank Masaru Hoshino (Kyoto University) for help with CD measurements. This work was supported
by JSPS KAKENHI (grant no. 16H04752), The Naito Foundation and the Suzuken Memorial Foundation to H.T.,
JSPS Fellowships to N.T., JSPS KAKENHI to M.S. and Health and Labor Science Research Grants for Research
from the Ministry of Health, Labor and Welfare to H.O., T.F. and Z.K.
N.T. and H.T. conceived of the project and wrote the manuscript. H.T. supervised the study. N.T., A.Y., T.K. and
H.T. performed experiments and analyzed data. T.K. and H.O. provided scientific insights. Z.K., T.F., M.S., H.O.
and H.T. raised funding and provided resources for the research. All authors edited or reviewed the manuscript.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42661-5.
Competing Interests: The authors declare no competing interests. Publisher?s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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