the stehlik lab

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regulation of cytosolic PRR signaling

ASC localization and splicing

 

The molecular mechanisms regulating cytosolic PRRs - and in particular inflammasome activity - are still poorly understood.  However, since excessive inflammasome activity is directly linked to a wide spectrum of inflammatory diseases, a better understanding of these control mechanisms is likely to provide a basis for the design of novel therapies.  We originally co-discovered that ASC is the essential adaptor for caspase-1 [1], and were the first to describe assembly of endogenous inflammasome components into cytosolic aggregates in macrophages and showed that this step requires the redistribution of the inflammasome adaptor from the nucleus to a perinuclear domain  [2].  We furthermore identified that differential splicing of ASC regulates inflammasome activation by producing proteins lacking key protein-protein interaction domains [3].

 

 

PYRIN domain-only proteins (POPs)

 

Inflammasome assembly is mediated by nucleation of the adaptor ASC by upstream PRRs and this step requires interaction via specific death domain fold-containing protein-protein interactions, namely the PYRIN domain (PYD) and caspase recruitment domain (CARD) (reviewed in [4, 5]).  We observed significant species differences in the repertoire of these proteins in humans and mice [6].  In particular, we discovered and established the family of small, single PYD-containing PYRIN domain-only proteins (POPs) [7], which are encoded in humans, but are lacking in mice [8].  Subsequently, we described all other full POP members, including POP1 [7, 9], POP2 [10, 11] and POP3 [12] in humans and also viral members [13] and demonstrated specificity of individual POPs for a particular inflammasome.  We demonstrated that POPs function by binding to the PYD in either ASC or upstream PRRs, occupying this essential protein interaction  motif and thereby preventing inflammasome assembly.

 

POP1 and POP2 bind to ASC and prevents recruitment to activated NLRP3. Consequently, caspase-1 activation impaired, which results in reduced release of inflammatory cytokines and reduced pyroptosis [9, 11].  POP3 on the other hand selectively binds to the ALRs AIM2 and IFI16 and prevents DNA and DNA virus-induced inflammasome activation and IL-18 production [12].  In addition, POP2 inhibits inflammasome priming [11].  As expected from key regulators, expression of POPs is undetected in  resting cells and highly inducible upon inflammatory activation of macrophages - in the case of POP1 by pro-inflammatory cytokines and PAMPs, such as LPS and DNA and type I interferons in the case of POP3.  Hence, POPs function in an inflammasome regulatory feedback loop and are likely involved in the resolution phase of inflammasome responses.

 

Since POPs are lacking from mice, we generated transgenic mice expressing POPs in the monocyte macrophage lineage and showed that POPs have a role in blocking excessive inflammasome responses in vivo [9, 11, 12].  In particular, POP1 and POP2 are capable of efficiently ameliorating inflammatory disease induced by PAMPs, such as LPS, by danger signals such as MSU crystals and auto inflammatory disease triggered by mutations in inflammasome components, such as NLRP3 mutations in Cryopyrinopathy [9, 11].

 

 

References

 

[1] Stehlik, C., Lee, S.H., Dorfleutner A., Stassinopoulos, A., Sagara, J., and Reed, J.C. (2003) Apoptosis-associated speck-like protein containing a caspase recruitment domain is a Regulator of pro-Caspase-1 Activation, J. Immunol. 171, 6154-63.

 

[2] Bryan, N.B., Dorfleutner, A., Rojanasakul, Y., and Stehlik, C. (2009) Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J. Immunol., 182, 3173-3182, PMC2652671. Featured “in this issue”.

 

[3] Bryan, N.B., Dorfleutner, A., Kramer, S.J., Yun, C., Rojanasakul, Y., and Stehlik, C. (2010) Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) regulates inflammasomes. J. Inflammation, 7, 23, PMC2887861. Highly Accessed Article

 

[4] Chu, L.H., Gangopadhyay, A., Dorfleutner, A. and Stehlik, C. (2015) An updated view on the structure and function of PYRIN domains. Apoptosis special issue on “The domains of apoptosis and inflammation”, Apoptosis, 20,157-173. PMC4297229.

 

[5] Dorfleutner, A. Chu, L. and Stehlik, C. (2015) Inhibiting the inflammasome: one domain at a time. Immunological Reviews, 265, 205-216. PMC4400809.

 

[6] Reed, J.C., Doctor, K., Rojas, A., Zapata, J.M., Stehlik, C., Fiorentino, L., Damiano, J., Roth, W., Matsuzawa, S., Newman, R., Takayama, S., Marusawa, H., Xu, F., Salvesen, G., and Godzik, A. (2003) Comparative Analysis of Apoptosis and Inflammation Genes of Mice and Humans. Genome Res.13(6B), 1376-1388.

 

[7] Stehlik, C., Krajewska, M., Welsh, K., Krajewski, S., Godzik, A., and Reed, J.C. (2003) The PAAD/PYRIN-only protein POP is a modulator of ASC-mediated NF-kB and pro-Caspase-1 regulation. Biochem. J. 372, 101-113. Highlighted by an editorial: Mariathasan, S., and Vucic, D. (2003) POPing the fire into the pyrin? Biochem. J. 372, e1-2.

 

[8] Stehlik, C. and Dorfleutner, A. (2007) COPs and POPs: Modulators of Inflammasome Activity. J. Immunol., 179, 7993-7998

 

[9] de Almeida, L., Khare, S., Misharin, A.V., Patel, R., Wallin, M., Perlman, H.R., Greaves, D.R., Hoffman, H.M., Dorfleutner, A., and Stehlik, C. (2015) The PYRIN domain-only protein POP1 inhibits inflammasome assembly and ameliorates inflammatory disease. Immunity, 43, 264-276. Selected for display at the Immunity website and highlighted with a Commentary by Shimada K. Timothy R. Crother T.R., and Arditi, M. (2015) POPsicle for Fever! Cooling Down the Inflammasome. Immunity, 43, 213-215.

 

[10] Dorfleutner, A., Bryan, N.B., Talbott, S.J., Fynya, K.N., Rellick, S.L., Reed, J.C., Shi, X., Royanasakul, Y., Flynn, D.C., and Stehlik, C. (2007) Cellular PYRIN domain-only protein (cPOP) 2 is a novel regulator of inflammasome activation. Infect. Immun., 75, 1484-1492, PMC1828547. Featured as a Spotlight article.

 

[11] Ratsimandresy, R., Chu, H.L., Indramohan, M., Gangopadhyay, A., Dorfleutner, A., and Stehlik, C. (2017) The PYRIN domain-only protein POP2 regulates inflammasome priming and activation. Nature Communications, 8, 15556.

 

[12] Khare, S., Ratsimandresy, R.A., de Almeida, L., Cuda, C.M., Rellick, S.L., Misharin, A.V., Wallin, M.C., Gangopadhyay, A., Forte, E., Gottwein, E., Perlman, H., Reed, J.C., Greaves, D.R., Dorfleutner, A. and Stehlik, C. (2014) The PYRIN domain-only protein POP3 inhibits AIM2-like receptor inflammasomes and regulates responses to DNA virus infections. Nature Immunology, 15, 343-345, PMC4123781. Selected as the cover article for the April issue and highlighted with a News and Views Commentary by Krishnaswamy J.K., Liu D. and Eisenbarth S.C. (2014) POP goes the inflammasome. Nat. Immunol., 15, 311-313

 

[13] Dorfleutner, A., Talbott, S.J., Bryan, N.B., Funya, K.N., Rellick, S.L., Reed, J.C., Shi, X., Flynn, D.C, Rojanasakul, Y. and Stehlik, C. (2007) A Shope Fibroma virus PYRIN-only protein modulates the host immune response. Virus Genes, 35, 685-694