Pyrin Domain-only Protein Research Articles

In vitro complex formation of human PYRIN domain-only protein 3 prevented by self-oligomerization of ASC PYD domain.

The formation of inflammasome complexes contributes inactivation of inflammatory caspases viz caspase 1, which is generally considered essential for the innate response. Three proteins constituted this inflammasome complex, such as Nod-like receptors (NLRP or AIM2), ASC possessing caspase-recruiting domain, and caspase-1. The ASC proteins comprise two domains, the N-terminal PYD domain responsible for the interaction of various proteins, including PYD only protein 3 (POP3), and the CARD domain for association with other proteins. The PYRIN Domain-Only Protein POP3 negatively regulates responses to DNA virus infection by preventing the ALR inflammasome formation. POP3 directly interacts with ASC, therefore inhibiting ASC recruitment to AIM2-like receptors (ALRs). In the current study, we designed various constructs of the PYRIN Domain-Only Protein 3 (POP3) and ASC PYD domain to find the best-overexpressed construct for biochemical characterization as well as our complex studies. We cloned, purified, and characterized the PYD domain of pyrin only protein 3 and ASC PYD domain under physiological conditions. Our in vitro study clearly shows that the ASC PYD domain of corresponding amino acid 1–96 aa with ease self-oligomerization in physiological buffer conditions, and complex formation of POP3 PYD (1–83 aa) was inhibited by ASC PYD domain. Besides, we purified the PYD of POP3 protein in low and high salt conditions and different pH values for their biochemical characterization. Our results showed that POP3 formed a dimer under normal physiological conditions and was stable under normal buffer conditions; however, the purification in extremely low pH (pH5.0) conditions shows unstable behavior, the high salt conditions (500 mM NaCl) influence the protein aggregation. SDS PAGE arbitrated the homogeneity of the PYD domain of pyrin only protein 3 and ASC PYD domain of corresponding amino acids 1–83 and 1–96, respectively. Furthermore, our native PAGE shows the PYD domain of pyrin; only protein 3 did not form a complex with ASC PYD domain because of oligomerization mediated by the PYD domain.

An Update on CARD Only Proteins (COPs) and PYD Only Proteins (POPs) as Inflammasome Regulators.

Inflammasomes are protein scaffolds required for the activation of caspase-1 and the subsequent release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death to restore homeostasis following infection and sterile tissue damage. However, excessive inflammasome activation also causes detrimental inflammatory disease. Therefore, extensive control mechanisms are necessary to prevent improper inflammasome responses and inflammatory disease. Inflammasomes are assembled by sequential nucleated polymerization of Pyrin domain (PYD) and caspase recruitment domain (CARD)-containing inflammasome components. Once polymerization is nucleated, this process proceeds in a self-perpetuating manner and represents a point of no return. Therefore, regulation of this key step is crucial for a controlled inflammasome response. Here, we provide an update on two single domain protein families containing either a PYD or a CARD, the PYD-only proteins (POPs) and CARD-only proteins (COPs), respectively. Their structure allows them to occupy and block access to key protein–protein interaction domains necessary for inflammasome assembly, thereby regulating the threshold of these nucleated polymerization events, and consequently, the inflammatory host response.

Inflammasomes and their regulation in periodontal disease: A review.

Interleukin-1β (IL-1β), which is secreted by host tissues leading to periodontal tissue inflammation, is a major pro-inflammatory cytokine in the pathogenesis of periodontal disease. The conversion of pro-IL-1β into its biologically active form is controlled by multiprotein complexes named as inflammasomes, which are key regulator of host defense mechanisms and inflammasome involved diseases, including the periodontal diseases. Inflammasomes are regulated by different proteins and processes, including pyrin domain (PYD)-only proteins (POPs), CARD-only proteins (COPs), tripartite motif family proteins (TRIMs), autophagy, and interferons. A review of in vitro, in vivo, and clinical data from these publications revealed that several inflammasomes including (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) have been found to be involved in periodontal disease pathogenesis. To the best of our knowledge, the current article provides the first review of the literature focusing on studies that evaluated both inflammasomes and their regulators in periodontal disease. An upregulation for inflammasomes and a downregulation of inflammasome regulator proteins including POPs, COPs, and TRIMs have been reported in periodontal disease. Although interferons (types I and II) and autophagy have been found to be involved in periodontal disease, their possible role in inflammasome activation has not evaluated yet. Modulating the excessive inflammatory response by the use of inflammasome regulators may have potential in the management of periodontal disease.

Differential expression of inflammasome regulatory transcripts in periodontal disease

The inflammasome modulates the release of key proinflammatory cytokines associated with periodontal disease pathogenesis. The aim of this study was to evaluate the expression of proteins that regulate the inflammasome, namely pyrin domain-only proteins (POPs), caspase activation recruitment domain (CARD)-only proteins, and tripartite motif-containing (TRIM) proteins, in periodontal diseases. A total of 68 participants (34 males and 34 females) were divided into four groups, including periodontal health (H), gingivitis (G), chronic periodontitis (CP), and aggressive periodontitis (AgP) based on clinical parameters. Gingival tissue samples were obtained from all participants for reverse transcription polymerase chain reaction (RT-PCR)-based gene expression analyses of molecules that regulate the inflammasome, including apoptosis-associated speck-like protein (ASC) containing CARD, caspase-1, interleukin-1β (IL-1β), interleukin-18 (IL-18), nucleotide-binding domain, leucine rich family (NLR) pyrin domain containing 3 (NLRP3), NLR family pyrin domain containing 2 (NLRP2), AIM2 (absent in melanoma 2), POP1, POP2, CARD16, CARD18, TRIM16, and TRIM20 by RT-PCR. NLRP3 and IL-1β were upregulated in the G, CP, and AgP groups compared with group H (P<0.05). AIM2 was downregulated in the CP group compared with the H, G, and AgP groups (P<0.05). TRIM20, TRIM16, and CARD18 were downregulated in the G, CP, and AgP groups compared with the H group (P<0.05). POP1 and POP2 were downregulated in the CP and AgP, and AgP and G groups, respectively (P<0.05). Active periodontal disease may result in downregulation of inflammasome regulators that may increase the activity of NLRP3 and IL-1β in periodontal disease.

Myxoma virus M013 protein antagonizes NF-κB and inflammasome pathways via distinct structural motifs

Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.

Pyrin-only protein 2 limits inflammation but improves protection against bacteria

Pyrin domain-only proteins (POPs) are recently evolved, primate-specific proteins demonstrated in vitro as negative regulators of inflammatory responses. However, their in vivo function is not understood. Of the four known POPs, only POP2 is reported to regulate NF-κB-dependent transcription and multiple inflammasomes. Here we use a transgenic mouse-expressing POP2 controlled by its endogenous human promotor to study the immunological functions of POP2. Despite having significantly reduced inflammatory cytokine responses to LPS and bacterial infection, POP2 transgenic mice are more resistant to bacterial infection than wild-type mice. In a pulmonary tularaemia model, POP2 enhances IFN-γ production, modulates neutrophil numbers, improves macrophage functions, increases bacterial control and diminishes lung pathology. Thus, unlike other POPs thought to diminish innate protection, POP2 reduces detrimental inflammation while preserving and enhancing protective immunity. Our findings suggest that POP2 acts as a high-order regulator balancing cellular function and inflammation with broad implications for inflammation-associated diseases and therapeutic intervention.

The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation

Inflammasomes are protein platforms linking recognition of microbe, pathogen-associated and damage-associated molecular patterns by cytosolic sensory proteins to caspase-1 activation. Caspase-1 promotes pyroptotic cell death and the maturation and secretion of interleukin (IL)-1β and IL-18, which trigger inflammatory responses to clear infections and initiate wound-healing; however, excessive responses cause inflammatory disease. Inflammasome assembly requires the PYRIN domain (PYD)-containing adaptor ASC, and depends on PYD–PYD interactions. Here we show that the PYD-only protein POP2 inhibits inflammasome assembly by binding to ASC and interfering with the recruitment of ASC to upstream sensors, which prevents caspase-1 activation and cytokine release. POP2 also impairs macrophage priming by inhibiting the activation of non-canonical IκB kinase ɛ and IκBα, and consequently protects from excessive inflammation and acute shock in vivo. Our findings advance our understanding of the complex regulatory mechanisms that maintain a balanced inflammatory response and highlight important differences between individual POP members.

POPsicle for Fever! Cooling Down the Inflammasome

Inhibition of the inflammasome might be beneficial for numerous inflammatory pathologies. In this issue of Immunity, de Almeida et al. (2015) report that the PYRIN domain-only protein (POP1) efficiently inhibits inflammasome activation, identifying it as a pan-inflammasome inhibitor.

Crystallization and preliminary X-ray crystallographic studies of cPOP1

Cellular pyrin domain-only protein 1 (cPOP1) is a pyrin domain (PYD)-containing protein that can regulate inflammation by preventing the assembly of inflammasome via direct interaction with ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain). In this study, cPOP1, corresponding to amino acids 1-87, was overexpressed in Escherichia coli using an engineered C-terminal polyhistidine tag. cPOP1 was then purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 3.6 Å from a crystal belonging to the cubic space group P2₁3 with unit-cell parameters a=b=c=94.12 Å, α=β=γ=90.00°.

Cellular pyrin domain-only protein 2 is a candidate regulator of inflammasome activation.

Pyrin domain (PYD) proteins have recently emerged as important signaling molecules involved in the development of innate immunity against intracellular pathogens through activation of inflammatory mediator pathways. ASC is the central adaptor protein, which links pathogen recognition by PYD-containing pathogen recognition receptors, known as PYD-Nod-like receptors (NLR), PAN, PYPAF, NALP, Nod, and Caterpiller proteins, to the activation of downstream effectors, including activation of caspase-1 and NF-kappaB. Activation of these effectors occurs when specific protein complexes, known as inflammasomes, are formed. PYD signal transduction leads to inflammasome assembly and activation of specific effector proteins. It is modulated by a cellular PYD-only protein (cPOP1), which binds to ASC and interferes with the recruitment of ASC to activated PYD-NLRs. Here we describe the identification and characterization of a second cellular POP (cPOP2), which shows highest homology to the PYD of PAN1. cPOP2 binds to ASC and PAN1, thereby blocking formation of cryopyrin and PAN1-containing inflammasomes, activation of caspase-1, and subsequent processing and secretion of bioactive interleukin-1beta. Existence of a second cPOP provides additional insights into inflammasome formation and suggests that POPs might be a common regulatory mechanism to "fine-tune" the activity of specific PYD-NLR family protein-containing inflammasomes.

Structure and Dynamics of ASC2, a Pyrin Domain-only Protein That Regulates Inflammatory Signaling

Structure and Dynamics of ASC2, a Pyrin Domain-only Protein That Regulates Inflammatory Signaling

Structure and Dynamics of ASC2, a Pyrin Domain-only Protein That Regulates Inflammatory Signaling

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-kappaB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha2-alpha3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha2-alpha3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha2-alpha3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.