7 research outputs found
Pyrin recruits PSTPIP1 to ASC specks.
<p>All images are from transfected COS cells. Representative images are shown. (A) The apoptotic speck protein, ASC (red), is normally diffusely distributed throughout the cell in cytoplasm and nucleus. (B) ASC (in this case, green) can coalesce into a small perinuclear aggregate, the speck. (C–D) Pyrin (red) is recruited to ASC specks via its PyD as previously shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone.0006147-Richards1" target="_blank">[32]</a>. (E–G) PSTPIP1 is not detected in ASC specks in the absence of co-transfected pyrin. (H–J) In 70% of cells transfected with untagged ASC, PSTPIP1-FLAG and pyrin-myc, PSTPIP1 is recruited to the speck (arrow, H). (K–P) In 30% of cases, transfection of the three proteins results in localization of pyrin in both PSTPIP1 filaments and in the speck (K–M), or exclusively in the speck (N–P). (Q–S) FLAG-tagged W232A PSTPIP1 does not interact with pyrin, and is not recruited to specks. (T–Y) Recruitment of PAPA mutants by myc-pyrin to the ASC speck. (T–V) A230T-FLAG. (W–Y) E250Q-FLAG. Pyrin recruits these mutant forms to ASC specks in 95% of transfected cells.</p
The B-box/coiled-coil region of pyrin is required for reticularization of PSTPIP1 filaments.
<p>Portions of pyrin's B-box and coiled-coil region (all myc tagged), were co-transfected with PSTPIP1-FLAG. (A–B) Pyrin exons 2–3 or (C–D) exons 4–5 do not bind PSTPIP1 filaments. Note that PSTPIP1 filaments (A,C) are generally straight. (E–F) Pyrin exons 2–4 decorates PSTPIP1 filaments, but does not alter their distribution. (G–I) The B-box and coiled-coil region of pyrin, encoded by exons 3–5, binds to and remodels PSTPIP1 filaments.</p
Patterns of pyrin and PSTPIP1 expression in native and transfected cells.
<p>(A) Immunostaining of native PSTPIP1 (green) in human monocytes reveals a finely branched pattern of filaments. (B) In human neutrophils, the PSTPIP1 distribution is filamentous and concentrated at the edge of the cell. (C) In transfected COS cells, PSTPIP1 forms long straight filamentous structures. (D) In human monocytes, pyrin (red) is distributed in a filamentous reticular pattern that extends throughout the cytoplasm and encircles the nucleus. (E) When epitope tagged pyrin (green) is transfected into COS cells, no reticular network is seen; pyrin is diffusely cytoplasmic.</p
Identification of the domains of PSTPIP1 that are required for filament formation and filament binding.
<p>(A) A schematic view of the PSTPIP1 molecule (416 amino acids) is shown at top. The F-BAR domain (∼300 amino acids) includes the FCH domain, an uncharacterized intervening region (X) and a coiled coil domain (CC). A short helical region (helix 5) of the F-BAR structure is contained within the 5′ end of an uncharacterized region (Y) downstream of the CC domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone.0006147-Henne1" target="_blank">[17]</a>. The SH3 domain includes amino acid residues 364–416. The location of three mutations associated with PAPA syndrome (at amino acids 230, 250 and 266) is shown; a mutation at position 232 abolishes PSTPIP1 binding to pyrin and to PTP HSCF, a PEST-type protein tyrosine phosphatase (PTP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone.0006147-Shoham1" target="_blank">[5]</a>. The specific deletion constructs tested are outlined in the lower schematic. Results of these studies are tabulated at right. “Forms filaments” indicates that the protein forms filamentous structures when transfected alone. “Binds filaments” means that the protein binds to formed full length filaments of co-transfected PSTPIP1. (B–J) Truncated versions of myc-tagged PSTPIP1 shown in (A) were transfected alone or in combination with full-length PSTPIP1-FLAG. B–E) The FCH and coiled-coil portion of PSTPIP1 bound filaments formed by full-length PSTPIP1 (B–D), but was not able to form filaments when transfected alone (E). (F,G) A PSTPIP protein containing the coiled-coil and SH3 region of PSTPIP1 was not able to form filaments (not shown), nor was it able to bind to filaments formed by full-length PSTPIP1 (G). (H) PSTPIP1 lacking the SH3 domain forms filaments when transfected alone. Thus, the SH3 domain is not required for filament formation. (I,J) The two PAPA-associated mutants, A230T (I) and E250Q (J) form long straight filaments similar to those of wildtype PSTPIP1.</p
PSTPIP1 filaments are membrane-associated.
<p>(A–C) DiI<sub>C16</sub> decorates PSTPIP1 filaments, indicating that the fibrils are membrane-associated. (D–F) In cells transfected with pyrin and PSTPIP1, pyrin produces the typical reticular pattern, and DiI<sub>C16</sub> staining is associated with the branched filaments. (G–I) In cells transfected with pyrin M694V-YFP and wild type PSTPIP1, filaments are visible and stain with DiI<sub>C16</sub>. (J–L) In cells transfected with wild type pyrin and PAPA-associated A230T-GFP, DiIC<sub>16</sub> filament staining is preserved.</p
FMF-causing mutations do not alter the appearance of reticular PSTPIP1 fibrils; PAPA-associated PSTPIP1 mutants are bound and reticulated by pyrin.
<p>(A–R) Myc-tagged versions of mutant pyrin were co-transfected with PSTPIP1-FLAG: (A–C) P369S; (D–F); E148Q; (G–I) M694V; (J–L) M680I; (M–O) V726A; (P–R) A744S. (S–X) PAPA syndrome-associated PSTPIP1 were co-transfected myc-pyrin: (S–U) A230T; (V–X) E250Q.</p
Co-expression of pyrin alters the distribution of PSTPIP1 in transfected cells.
<p>(A–E) In cells co-transfected with myc-tagged pyrin and FLAG-tagged PSTPIP1, PSTPIP1 filaments are branched and reticulated, and pyrin co-localizes with these filaments. (A) and (C) illustrate the original images, while (B) and (D) are processed images that have been deconvoluted to remove background and enhance the signal of the filaments. A branched network of filaments surrounding the nucleus is evident. (E) Overlay of pyrin and PSTPIP1 staining pattern after deconvolution; pyrin appears to be concentrated at the nodes of branch points. (F–H) FLAG-tagged PSTPIP1 lacking the SH3 domain, PSTPIP1(-SH3), can recruit myc-tagged pyrin to filaments. Pyrin binding causes the filaments to be highly branched or reticular (compare the PSTPIP1 pattern in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone-0006147-g004" target="_blank">Figure 4G</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone-0006147-g002" target="_blank">Figure 2H</a>). (I–K) The W232A mutation of PSTPIP1, which cannot bind pyrin, forms straight filaments (green, J), and pyrin (red, I) does not decorate or reticularize these filaments. The overlay is shown in (K). Note that though filaments appear yellow, there is no filamentous pattern of pyrin (I); rather, pyrin is uniformly distributed.</p