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. 2016 Mar 8;113(10):2720-5.
doi: 10.1073/pnas.1522361113. Epub 2016 Feb 22.

Identification of a Novel Cell Death-Inducing Domain Reveals That Fungal Amyloid-Controlled Programmed Cell Death Is Related to Necroptosis

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Free PMC article

Identification of a Novel Cell Death-Inducing Domain Reveals That Fungal Amyloid-Controlled Programmed Cell Death Is Related to Necroptosis

Asen Daskalov et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a β-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis.

Keywords: amyloid; incompatibility; necroptosis; prion; programmed cell death.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The PP motif gene cluster. (A) Representation of the CHGG_1412/13/14 locus with three adjacent genes, one encoding a HELL domain protein termed HELLP, one encoding a predicted lipase related to SesB (SBP), and one encoding a NB-ARC/TPR STAND protein (PNT1). The PP motif is represented as a pink line. (B) Alignment of the PP motif region of HELLP, SBP, and PNT1 together with a portion of the RHIM motif of human RIP1 and RIP3. (C) Sequence signatures for the central region of PP and the RHIM motif generated with Skylign. Letter size corresponds to level of conservation.
Fig. S1.
Fig. S1.
Conservation of a predicted N-terminal transmembrane helix in HELLP. (A) Prediction of a transmembrane helix for HELLP and HET-S using TMHMM. The probability of transmembrane helix formation for N-terminal residues is given. (B) Alignment of the N-terminal regions of HeLo and HeLo-like domain proteins. HET-S and HELLP and the eight strongest homologs of each protein identified in the National Center for Biotechnology Information’s nr database were aligned. HELLP and HET-S are boxed in red and blue, respectively. The G9 and G13 residues of HELLP are boxed in black.
Fig. 2.
Fig. 2.
HELLP(172-278)-RFP forms a prion, incompatible with full-length HELLP. (A) Micrograph of a P. anserina strain expressing a HELLP(172-278)-RFP fusion protein in either the soluble [π*] state (Upper) or the aggregated [π] state (Lower). (Scale bar: 5 μ.) (B) Confrontation of a strain expressing full-length HELLP and expressing HELLP(172-278)-RFP in the soluble state [π*] or aggregated state [π] as noted. Note the formation of a barrage (marked by a white arrow) specifically in the confrontation with the strain expressing HELLP(172-278)-RFP in the aggregated state [π].
Fig. S2.
Fig. S2.
Expression of HELLP(215-278)-GFP and prion propagation of HELLP(172-278)-RFP. (A) Strains expressing HELLP(215-278)-GFP from a barrage reaction (marked by white arrows) when confronted to strains expressing full-length HELLP (HELLP-RFP). The control was an untransformed ΔPa_5_8070 strain. (B) HELLP(215-278)-GFP forms aggregates when expressed in P. anserina. (Scale bar: 5 μ.) (C) Lack of cross-infection between [π] and [Het-s] prions. The same picture is shown with and without labeling for clarity. (Right) Strains were confronted on solid medium. [π] strains produce a barrage to strains expressing HELLP (white arrowheads), and [Het-s] strains (s) produce a barrage to [Het-S] strains (S) (gray arrowheads). On confrontation with a [π] strain, a [π*] strain acquires the [π] phenotype and produces a barrage to HELLP (red arrowhead). A [Het-s*] strain (s*) confronted to a [Het-s] acquires the [Het-s] phenotype and produces a barrage to [Het-S] (black arrowhead); however, [Het-s] strains do not convert [π*], and [π] strains do not convert [Het-s*].
Fig. 3.
Fig. 3.
HELLP-RFP localizes at the cell periphery on interaction with aggregated HELLP(215-278)-GFP. On fusion of cells coexpressing HELLP-RFP and HELLP(215-278)-GFP under the aggregated state [π], HELLP-RFP relocalizes to the membrane region. The white arrowhead marks the position of the cell fusion point between the HELLP-RFP and HELLP(215-278)-GFP strains. (Scale bar: 5 μ.)
Fig. S3.
Fig. S3.
Localization of HELLP-RFP in homokaryotic mycelium and localization of HELLP-RFP point mutants. (A) HELLP-RFP is expressed in a wild-type strain and shows a diffuse cytoplasmic localization. (Scale bar: 5 μ.) (B) HELLP-RFP G9I and G13I mutants do not produce a barrage reaction to HELLP(215-278)-GFP, whereas wild-type HELLP-RFP does (white arrowhead). (C) Localization of HELLP-RFP G9I and HELLP-RFP G13I in fusion cells with strains expressing HELLP(215-278)-GFP. (Scale bar: 5 μ.)
Fig. S4.
Fig. S4.
HELLP(215-278), SBP(219-280), and PNT1(23-38) form amyloid fibrils. (A) Electron micrograph of negatively stained HELLP(215-278) fibrils. (Scale bar: 30 nm.) (B) X-ray diffraction of HELLP(215-278) fibrils. Note the major diffraction at 4.7 Å. (C) FTIR analysis of HELLP(215-278) fibrils showing a major band at 1,630 cm−1 indicative of the presence of parallel stacked β-sheets. (D) Electron micrograph of HELLP(215-278) fibrils formed in 8 M urea. (Scale bar: 100 nm.) (E) Electron micrograph of negatively stained SBP(219-280) fibrils. (Scale bar: 30 nm.) (F) FTIR analysis of SBP(219-280) fibrils showing a major band at 1,630 cm−1. (G) Sequence and alignment of three 26-aa peptides related to the PP motif and analyzed by EM. (H) Electron micrograph of the assemblies formed by the synthetic peptide as given in E. (Scale bar: 100 nm.)
Fig. S5.
Fig. S5.
Proteinase K-resistant core of HELLP(210-278) and SBP(219-280) fibrils. (A) SDS/PAGE analysis of HELLP(215-278) fibrils after proteinase K digestion. Fibrils were digested for the indicated times. (B) Secondary structure prediction for HELLP and SBP using PSIPRED. The proteinase K-protected region as identified by mass spectrometry is boxed in red. The position of the PP motif is underlined in red. The beginning of the proteinase K-resistant region extends into the last predicted α-helix of the HELL domain. Note that the electrophoretic mobility of the peptides on SDS/PAGE (in A) is slightly aberrant (with an apparent mass of ∼7 kDa), possibly owing to the high content in charged residues in the N-terminal part of the protected region. (C) Alignment of the proteinase K-resistant regions of HELLP and SBP fibrils.
Fig. 4.
Fig. 4.
Conformational analysis of HELLP(215-278) fibrils by solid-state NMR. (A) 1D 13C-detected CP spectrum. (B) 2D 1H-13C INEPT spectrum. (C) Excerpt of 2D PDSD ssNMR 13C-13C spectra centered around the Thr resonance frequency (Upper, 30 ms mixing time; Lower, 150 ms mixing time), optimized to detect intraresidual (30 ms) and sequential (150 ms) 13C-13C correlations. All peak assignments of the Thr271 spin system except for the diagonal Cα-Cα peaks are annotated. The additional peaks visible in the red spectrum reveal interresidual contacts of the Thr spin system. Assignments of Gly270 and Met272 are annotated above the spectral excerpt. The complete aliphatic spectral region of the 2D PDSD (30 ms mixing time) is shown in Fig. S5D. (D) Secondary ssNMR chemical shifts ΔδCα-ΔδCβ of the Gly270-Thr271-Met272 amino acid stretch in HELLP(215-278) (black), revealing their β-strand secondary structure. The secondary chemical shifts of a Gly-Thr-Met stretch in typical α-helical and β-strand conformations (blue and orange, respectively) are plotted for eye guidance. (E) 1D trace of the 2D PDSD (150 ms mixing time) at the position of the dotted blue line indicated in C, Lower. The line widths of intense signals are denoted.
Fig. S6.
Fig. S6.
Solid-state NMR PDSD spectrum of HELLP(215-278) fibrils, recorded at 300-MHz 1H frequency with an MAS rate of 11 kHz.
Fig. S7.
Fig. S7.
Expression of SBP-GFP in P. anserine. (A) P. anserina strains expressing SBP-GFP or SBP(219-280)-GFP and showing dot formation. (Scale bar: 5 μ.) (B) Strains expressing SBP-GFP produce a barrage reaction to strains expressing HELLP-RFP (white arrowheads). The control strain is an untransformed ΔPa_5_8070 strain. (C) In fusion cells between strains expressing HELLP-RFP and SBP-GFP and displaying SBP-GFP aggregates, SBP and HELLP colocalize in the plasma membrane region. The recipient strain background is ΔPa_5_8070. The arrows point to the presumed fusion points between the two strains. (Scale bar: 5 μ.)
Fig. 5.
Fig. 5.
Homology between the HELL domain and the pore-forming domain of MLKL. Shown is alignment of the HELL domain of HELLP (and various fungal homologs) with the 4HB domain region of various MLKL homologs from different phylogenetically diverse chordate species. The C. globosum HELLP sequence is boxed in orange, and the human MLKL sequence is boxed in red. The secondary structure of human MLKL (after PDB ID code Q8NB16) is given below the alignment. Alignment was generated with MAFFT with default settings.
Fig. S8.
Fig. S8.
Alignment of chordate MLKL homologs, plant RPW8 domain proteins, and fungal HeLo-like domain proteins. Phylogenetically diverse chordate MLKL homologs, plant RPW8 proteins, and fungal HeLo-like domain protein sequences were in alignment with MAFFT with default settings. The HELLP, mouse MLKL, and Arabidopsis RPW8.1 sequences are boxed in orange, red, and green, respectively. Conserved negatively charged residues at position +2 in MLKL homologs are boxed in purple.

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