Somatic Mutations in NEK9 Cause Nevus Comedonicus

Acne vulgaris (AV) affects most adolescents, and of those affected, moderate to severe disease occurs in 20%. Comedones, follicular plugs consisting of desquamated keratinocytes and sebum, are central to its pathogenesis. Despite high heritability in first-degree relatives, AV genetic determinants remain incompletely understood. We therefore employed whole-exome sequencing (WES) in nevus comedonicus (NC), a rare disorder that features comedones and inflammatory acne cysts in localized, linear configurations. WES identified somatic NEK9 mutations, each affecting highly conserved residues within its kinase or RCC1 domains, in affected tissue of three out of three NC-affected subjects. All mutations are gain of function, resulting in increased phosphorylation at Thr210, a hallmark of NEK9 kinase activation. We found that comedo formation in NC is marked by loss of follicular differentiation markers, expansion of keratin-15-positive cells from localization within the bulge to the entire sub-bulge follicle and cyst, and ectopic expression of keratin 10, a marker of interfollicular differentiation not present in normal follicles. These findings suggest that NEK9 mutations in NC disrupt normal follicular differentiation and identify NEK9 as a potential regulator of follicular homeostasis

Anthrax Lethal Factor Cleavage of Nlrp1 Is Required for Activation of the Inflammasome

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT

Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both Toxin-Sensitive and Toxin-Resistant Macrophages

<div><p>Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.</p> </div

Caspase-1 activation in bone marrow-derived mouse macrophages.

<p>LPS-primed (1 µg/ml, 2 h) bone marrow-derived macrophages from Balb/cJ or NOD/LtJ mice were treated with LT (1 µg/ml) for 60 or 80 min, or with nigericin at indicated doses for 20 min. Cell lysates were analyzed by Western blotting for IL-1β, and the same samples were probed with caspase-1 p10 antibody to detect caspase-1 cleavage.</p

Cleavage of full length rat and mouse Nlrp1b proteins by LF.

<p>(A) IP (anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged mouse Nlrp1b (BALB) or rat Nlrp1(CDF) proteins following treatment with LF (1 µg/ml) for 15 min or 2 h. Cleavage of CDF Nlrp1 leads to appearance of a 6-kDa HA-reactive band and cleavage of BALB Nlrp1b leads to a slightly smaller fragment. (B) IP (anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged Nlrp1b proteins or control vector following treatment with LF (1 µg/ml, 30 min). Anti-HA cross-reactive bands not marked as HA-Nlrp1 also appear in vector-transfected controls. (C) Comparison of size of cleavage fragments generated after cleavage of BALB and NOD HA-tagged Nlrp1b (using conditions same as 2B), indicating the smaller size of the fragment generated following cleavage of the BALB protein (Western representative of five similar experiments).</p

Cleavage of mouse BALB118 and NOD118 Nlrp1 fusion proteins by LF.

<p>(A, B) <i>In vitro</i> cleavage of N-terminally 6His-GST-tagged aa 3-118 of Nlrp1b proteins. Purified proteins (0.53 mg/ml or 0.94 mg/ml, in A and B, respectively) were treated with the indicated molar ratios of LF, or with a 1∶10 molar ratio of the mutant LF E687C (LFm), for 4 h prior to SDS gel electrophoresis and Coomasie staining. F1 and F2 refer to two fragments generated following LF treatment. (C) GST-tagged or double alanine mutant variants (0.44-0.66 mg/ml) were treated with 33 µg/ml LF or LFm for 4 h prior to SDS gel electrophoresis and Coomassie staining.</p

Nlrp1 protein alignments and constructs.

<p>(A). Alignment of amino acid sequences from the N-terminus of mouse Nlrp1b and rat Nlrp1 proteins. Sequences shown are those of 4 mouse and 2 rat strains, including strains having macrophages that are either sensitive (S) or resistant (R) to LT. The previously identified LT cleavage site after residue 44 in rat CDF Nlrp1 is indicated by an arrow. The red box indicates the region of mouse sequence shown in (B). (B) Nlrp1b constructs used in this study with focus on N-terminal regions containing putative LF cleavage sites. The top two constructs represent the full-length HA-tagged Nlrp1b proteins from the LT-sensitive Balb/cJ (BALB) and the LT-resistant NOD/LtJ (NOD) macrophages, which were expressed in HT1080 cells. Full length NOD Nlrp1b is shorter (1172 aa) than BALB Nlrp1b due to a region downstream of the leucine rich repeat domain that is missing in this protein. The next four constructs represent proteins where aa 3-118 of Nlrp1b were expressed and purified from <i>E. coli</i> as N-terminal GST-tagged proteins. These proteins also contain a C-terminal His6 tag (not represented in figure). In the sequence alignments, residues identical to those in the construct listed above are indicated by quotation marks (“). Putative LF cleavage sites based on previously described motifs are drawn as vertical dotted lines below filled arrows. The MEK4 cleavage site is also aligned with both putative Nlrp1b cleavage sites. The last two sequences are those of constructs having two key lysine residues substituted with alanine.</p

Cleavage of full length rNlrp1 by LF.

<p>(A) Treatment of stably-transfected HT1080 cells overexpressing HA-tagged rNlrp1 proteins with LT (1 µg/ml) for 5 h followed by lysis and Western blotting with anti-HA and anti-Mek3 antibodies. Full length and cleaved HA-tagged rNlrp1 are shown. The central panel shows full length and cleaved MEK3 (in a reprobing of the same gel). Cleavage of rNlrp1 leads to appearance of the 6-kDa HA-reactive band. Cleavage of endogenous MEK3 leads to a shift in mobility. (B) Sucrose lysates from stably-transfected HT1080 cells overexpressing HA-tagged rNlrp1 proteins were treated with LF (1 or 10 µg/ml) for 3 h, and subjected to Western blotting with anti-HA (top row) or anti-MEK3 antibody (second row). Cleavage of rNlrp1 leads to loss of the N-terminal HA epitope, and cleavage of endogenous MEK3 leads to a shift in mobility. (C) IP (anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged rNlrp1 following treatment with LF (10 µg/ml, 3.5 h). Anti-HA reactive cross-reactive bands not marked as HA-Nlrp1 also appear in vector-transfected controls (data not shown).</p

Cleavage of CDF100 by LF.

<p>(A) <i>In vitro</i> cleavage of N-terminally 6His-GST-tagged aa 3–100 of rNlrp1^S (CDF100). Purified protein (1 mg/ml) was treated with the indicated molar ratios of LF for 6 h prior to SDS gel electrophoresis and Coomasie staining. (B) 6His-GST-tagged CDF100 protein or variants (1 mg/ml) were treated with recombinant LF or enzymatically inactive LF E687C (each at 128 µg/ml) for 3 h prior to SDS gel electrophoresis and Coomasie staining. F1 and F2 refer to two fragments generated following LF treatment of CDF100 and its mutated variants.</p

LT effects on cells expressing rNlrp1 constructs.

<p>(A) BMAJ cells (normally resistant to LT) that were retrovirally transfected with the indicated rNlrp1 constructs were treated with LT (PA+LF) for 10 h. Percent viability was assessed by MTT staining and calculated relative to untreated controls. Graph shown is from a single experiment, which is representative of eight identically performed experiments. Each point represents the average of three wells. Insets show staining of representative sensitive and resistant cell lines following toxin treatment. (B) Supernatant levels of IL-1β measured by ELISA. Cells were pretreated for 2 h with LPS (0.5–1 µg/ml), prior to LT treatment (7 h). Results shown are from a single experiment, which is representative of three similar experiments. Each point represents the average of duplicate wells in this experiment. (C) Protection of the sensitized BMAJ cell line transfected with rNlrp1fusion CDF53-LEW against challenge with LT (5 µg/ml) following treatment with 50 µM of each drug, or heat shock. Results are average of three experiments, with duplicate wells per treatment in each experiment. (D) Average supernatant IL-1β levels associated with protective treatments from (C) are shown. Results are the average of three experiments, with duplicate wells per treatment in each experiment.</p