Journal: Biochemical and biophysical research communications

Article Title: Cold-induced loss of interaction with HSC70 triggers inflammasome activity of familial cold autoinflammatory syndrome-causing mutants of NLRP3.

doi: 10.1016/j.bbrc.2022.12.018

Figure Lengend Snippet: Fig. 1. HSC70 complexes with cellular NLRP3 and shows enhanced interaction with FCAS-associated NLRP3 mutants. (A) Schematic showing domain organization of NLRP3. Sites of FCAS1-associated mutations in NLRP3 are indicated. PYD, pyrin domain; NBD, nucleotide-binding domain; LRR, leucine-rich repeat domain. (B) Endogenous NLRP3 forms a complex with HSC70. Western blot analysis of immunoprecipitates of NLRP3 obtained from lysates of differentiated THP1 cells shows interaction between NLRP3 and HSC70. Lysate was subjected to immunoprecipitation using NLRP3 antibody (IP: NLRP3) or normal IgG (Con IgG). WCL, whole cell lysate. GAPDH was used as a control to check for any non-specific binding. (C) Lysates of HEK293T cells transiently expressing GFP-tagged NLRP3 and its mutants were subjected to immunoprecipitation using GFP antibody. Western blot analysis showed presence of HSC70 in the immunoprecipitates (IP) of WT-NLRP3 as well as R260W and L353P mutants. (D) Bar diagram shows quantitation of relative abundance of HSC70 in IP of R260W and L353P mutants compared to WT-NLRP3 normalized with GFP signal from 5 independent experiments; n ¼ 5. *p < 0.05.

Article Snippet: Expression vectors- NLRP3 expression vectors pEGFP-C2-NLRP3 (#73955) and pCDNA-Myc-NLRP3-R260W (#73956) were obtained from Addgene, USA.

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Control, Expressing, Quantitation Assay

Journal: Biochemical and biophysical research communications

Article Title: Cold-induced loss of interaction with HSC70 triggers inflammasome activity of familial cold autoinflammatory syndrome-causing mutants of NLRP3.

doi: 10.1016/j.bbrc.2022.12.018

Figure Lengend Snippet: Fig. 2. HSC70 negatively regulates inflammasome formation by NLRP3 mutants. (A) HEK293T cells were transfected with HSC70-specific siRNA or control siRNA (Con siRNA), and after 42h of transfection, siRNA mediated knockdown of HSC70 was assessed by western blotting. GAPDH was used as loading control. (B) Effect of HSC70 knockdown on speck formation by WT-NLRP3, NLRP3-R260W and NLRP3-L353P. Representative images show enhanced ASC-speck formation by NLRP3 mutants upon siRNA mediated HSC70 knockdown. Cells co-expressing HA-ASC and Myc-NLRP3, Myc-NLRP3-R260W or Myc-NLRP3- L353P were scored for presence of specks. ASC-specks formed inside the cells are indicated by white arrowheads. Scale bars, 10 mm. (C) Bar diagram shows quantitation of effect of HSC70 knockdown on ASC-speck formation by NLRP3, NLRP3-R260W and NLRP3-L353P. n ¼ 3. **p < 0.005.

Article Snippet: Expression vectors- NLRP3 expression vectors pEGFP-C2-NLRP3 (#73955) and pCDNA-Myc-NLRP3-R260W (#73956) were obtained from Addgene, USA.

Techniques: Transfection, Control, Knockdown, Western Blot, Expressing, Quantitation Assay

Journal: Biochemical and biophysical research communications

Article Title: Cold-induced loss of interaction with HSC70 triggers inflammasome activity of familial cold autoinflammatory syndrome-causing mutants of NLRP3.

doi: 10.1016/j.bbrc.2022.12.018

Figure Lengend Snippet: Fig. 3. Effect of subnormal temperature on interaction of NLRP3 and its FCAS-associated mutants with HSC70. (A) HEK293T cells expressing GFP constructs of indicated plasmids were grown at 37 C for 16h or exposed to 28 C for 4h after 12h of transfection. Cell lysates were subjected to immunoprecipitation using GFP antibody and immunoprecipitates analyzed by western blotting. WT, wild-type NLRP3. (B) Quantitation of co-precipitated HSC70 at 37 C and at 28 C is shown. Values are normalized with corresponding IP signals. n ¼ 4. *p < 0.05; **p < 0.005. (C) THP1 cells were treated with 10 nM PMA for 72h at 37 C, or exposed to 28 C for 6h after 66h of treatment with PMA. Cell lysates were subjected to immunoprecipitation using NLRP3 antibody (IP) or normal IgG (Con) and immunoprecipitates analyzed by western blotting. (D) Bar diagram shows quantitation of relative binding of HSC70 with NLRP3 at 37 C and 28 C. n ¼ 3. *p < 0.05.

Article Snippet: Expression vectors- NLRP3 expression vectors pEGFP-C2-NLRP3 (#73955) and pCDNA-Myc-NLRP3-R260W (#73956) were obtained from Addgene, USA.

Techniques: Expressing, Construct, Transfection, Immunoprecipitation, Western Blot, Quantitation Assay, Binding Assay

Journal: Biochemical and biophysical research communications

Article Title: Cold-induced loss of interaction with HSC70 triggers inflammasome activity of familial cold autoinflammatory syndrome-causing mutants of NLRP3.

doi: 10.1016/j.bbrc.2022.12.018

Figure Lengend Snippet: Fig. 4. Effect of subnormal temperature on inflammasome formation and caspase-1 activation by NLRP3 mutants. (A) HEK293T cells coexpressing HA-ASC along with wildtype NLRP3, NLRP3-R260W or NLRP-L353P were scored for presence of ASC-specks. For cold-exposure, one set of cells was shifted to 28 C for 4h after 12h of transfection while the other set was maintained at 37 C. Representative immunofluorescence images show increase in percentage of cells forming ASC-specks upon exposure to subnormal temperature. White arrowheads indicate specks. Scale bars, 10 mm. (B) Bar diagram shows quantitation of ASC-speck formation upon exposure of cells to subnormal temperature. n ¼ 6. ***p < 0.0005. (C) Western blot analysis shows enhanced IL-1b maturation by NLRP3-R260W and NLRP-L353P mutants upon exposure of cells to 28 C. HEK293T cells were transfected with indicated NLRP3 plasmids along with caspase-1, ASC and pro-IL-1b. The transfected cells were grown at 37 C for 24h or incubated at 28 C for 6h after 18h of transfection. (D) Bar diagram shows quantitation of relative abundance to mature IL-1b (p17) normalized with the levels of pro-IL-1b (p32). n ¼ 5. *p < 0.05.

Article Snippet: Expression vectors- NLRP3 expression vectors pEGFP-C2-NLRP3 (#73955) and pCDNA-Myc-NLRP3-R260W (#73956) were obtained from Addgene, USA.

Techniques: Activation Assay, Transfection, Quantitation Assay, Western Blot, Incubation

Journal: Biochemical and biophysical research communications

Article Title: Cold-induced loss of interaction with HSC70 triggers inflammasome activity of familial cold autoinflammatory syndrome-causing mutants of NLRP3.

doi: 10.1016/j.bbrc.2022.12.018

Figure Lengend Snippet: Fig. 5. A model showing proposed mechanism of regulation of FCAS-causing mutants of NLRP3 by HSC70 in a temperature-dependent manner. Wild-type NLRP3 interacts with HSC70 and this interaction is increased by FCAS-causing mutations of NLRP3. It is proposed that FCAS-causing mutations induce a conformational change in NLRP3 that exposes HSC70-binding sites resulting in enhanced interaction of these mutants with HSC70. This interaction with HSC70 keeps the activity of NLRP3 mutants suppressed. Upon exposure to subnormal temperature HSC70 undergoes a conformational change resulting in loss of binding to the NLRP3 mutants. This leads to enhanced inflammasome formation and caspase- 1 activation by the mutants resulting in enhanced cytokine maturation and release.

Article Snippet: Expression vectors- NLRP3 expression vectors pEGFP-C2-NLRP3 (#73955) and pCDNA-Myc-NLRP3-R260W (#73956) were obtained from Addgene, USA.

Techniques: Binding Assay, Activity Assay, Activation Assay