Part 3: The PYHIN protein family

December 19, 2016

PYHIN proteins, also known as the HIN200 family, are characterized by the presence of Pyrin and HIN domains in their structure [6]. The N-terminal PYRIN (PYD) domain, through homotypic interactions, recruits proteins for the formation of complexes, such as inflammasomes, but these proteins are also involved in apoptosis, inflammation and cell cycle processes. The HIN domain is characteristic of the PYHIN family and localized at the C-terminal of the proteins, with the specific function of binding DNA. This 200 amino acid domain has been classified in three subtypes (A, B and C) based on sequence similarity.

There are five human PYHIN members: Absent in melanoma 2 (AIM2), Myeloid cell nuclear differentiation antigen (MNDA), IFN-γ inducible protein 16 (IFI16), Pyrin and HIN domain family member 1 (PYHIN-1) and Pyrin domain only protein 3 (POP3) (see Figure 4). These proteins were originally classified as important regulators of several functions such as cell cycle control, tumor suppression and transcriptional regulation, however further studies highlighted their role in the immune response during viral infections. Two members, AIM2 and IFI16, were then gathered in a new class of PRRs called AIM2-like receptors (ALRs), intracellular innate immune sensors which detect microbial DNA. PYHINs have different localization in the cell compartments, in particular, IFI16, MNDA and PYHIN-1 are usually found in the nucleus due to the presence of an N-terminal nuclear localization sequences (NLS), whereas AIM2 and POP3 are found in the cytoplasm [6].




Figure 4: Structure of the human PYHINs



AIM2

AIM2 is a cytosolic protein initially described as a tumor suppressor and is inducible by IFNγ, as are MNDA and IFI16. Its role in innate immunity is related with the ability of its HIN C domain to detect the presence of cytosolic non self-dsDNA, such as poly(dA:dT), leading to the multi-protein inflammasome complex formation where Caspase 1 is activated and pro-IL-1β and pro-IL-18 are processed to their mature forms. Human AIM2 and its murine ortholog can detect many pathogens, such as Vaccinia virus (VACV), mouse cytomegalovirus (MCMV), Francisella tularensis and Listeria monocytogenes. Moreover, human AIM2 activity can be inhibited by the interaction with another two proteins of the same family, IFI16 and POP3. Reduction in AIM2 levels were reported in colorectal and prostate cancer, suggesting a protective role of AIM2 in the development of tumorigenic diseases.

IFI16
Three isoforms (a, b, c), derived by alternative RNA splicing, compose the cluster of IFI16 protein variants. Initially IFI16 was identified as an IFNγ-inducible protein, constitutively expressed in many human lymphoid cell lines, with a role in cell cycle control and regulation of gene transcription events. IFI16 enhances the transcriptional activity of p53, a tumor suppressor protein, by binding its C-terminus. IFI16 is also able to bind BRCA1 and the retinoblastoma tumor-suppressor protein Rb showing an active role in apoptosis and transcriptional repression respectively. IFI16 was also proposed as a novel DNA sensor by Unterholzner et al through the detection of a 70 base pair IFN-β inducing Vaccinia virus dsDNA motif in the cytosol, and potentially signalling via the adaptor protein STING.

IFI16 has a nuclear localization sequence (NLS) and also nuclear export signal (NES) sequence which allow this immune sensing protein to recognize microbial DNA in both the nucleus and cytoplasm depending on the acetylation status of lysine residues at positions 99 and 128. This event leads to IFI16 acetylation and its translocation to the cytosol with the induction of innate responses such as inflammasome assembly (with the production of IL-1β) and IFI16-STING interaction (which causes IRF-3 phosphorylation, nuclear translocation of pIRF-3 and type I IFN production). Downstream signalling from IFI16 to STING takes place in the cytosol following either nuclear or cytosolic DNA recognition. However, the exact mechanism of how IFI16 activates STING is still unclear despite the recent discovery of the cGAS-cGAMP pathway, which IFI16 may cooperate with to activate STING.

MNDA

Myeloid cell Nuclear Differentiation Antigen (MNDA) is a protein which contains an N-terminal pyrin domain and a C-terminal HIN A domain. In 1998, it was the first human member of the PYHIN family to be characterized in the leukemia HL-60 cell line with a specific role in myeloid differentiation. It is predominantly found in the nuclear compartment and can actively bind dsDNA, but at different sites compared to those used by the HIN C domain of AIM2 and the HIN B domain of IFI16 [165]. MNDA has also been proposed as a regulator of gene transcription and consequently gene expression.

POP3
The most recently discovered member of the PYHIN family is POP3, a cytosolic protein of 114 amino acids with its gene localized between PYHIN1 and IFI16. This protein contains a PYD domain but lacks HIN domains, therefore POP3 is not able to bind DNA molecules. It has also been shown how its pyrin domain can interact with PYDs of IFI16 and AIM2 but not PYHIN1 and MNDA, thus negatively regulating inflammasome formation and IL-1β release mediated by the two members of the ALR family, IFI16 and AIM2 [6].

PYHIN-1
PYHIN-1, also known as IFIX, is the fifth member of the 200-amino-acid repeat (HIN-200) family. Its gene is predicted to code for six different isoforms by alternative splicing, α1, α2, β1, β2, ɣ1 and ɣ2, which all share a common N-terminal region with a PYD and a NLS; therefore, these proteins are localized in the nuclear compartment.

PYHIN-1 proteins were initially linked with a role in transcriptional regulation for genes involved in cell growth, differentiation and apoptosis [6]. A recent meta-analysis of genome-wide association studies correlated single nucleotide polymorphisms (SNPs) present in the PYHIN-1 gene region with asthma in African-American and African-Caribbean populations but not in Latinos or European Americans, suggesting a possible link between this PYHIN protein and asthma pathogenesis. However, further analysis conducted by Kantor et al. did not confirm the significant linkage between asthma-associated loci and PYHIN-1 gene in individuals of African ancestry.

In 2015, a new paper was published by Diner et al. suggesting that PYHIN-1 is a sensor of viral DNA [7]. First, they confirmed PYHIN-1s predominant nuclear localization within nucleoli. Second, according to a cell-based system which characterizes the interactome of PYHIN proteins, PYHIN-1 was shown to interact with proteins involved in dsDNA repair, telomerase maintenance and chromatin remodeling, similar to AIM2, but also with proteins involved in apoptosis, antiviral defence and transcription. Third, based on PYHIN-1:PML interaction and with the fact that PYHIN-1 is a member of the immune regulator PYHIN family, further analysis have been conducted in order to evaluate its possible antiviral activity. The knockdown of PYHIN-1 in fibroblast cells, using shRNAs targeting all six isoforms, significantly enhanced HSV-1 titers compared to control cells, suggesting that PYHIN-1 inhibits HSV-1 replication; the same effect was also observed in PML-depleted fibroblasts.

Diner et al. also proposed that PYHIN-1 directly participates in the activation of type I IFN at transcriptional level in the nuclear compartment, according to the functional interactome results where the protein was able to interact with members of chromatin remodeling complexes and transcriptional regulators, similar to IFI16 [7].





Figure 5: PYHINs and DNA sensing


[1] J. Parkin and B. Cohen. An overview of the immune system. Lancet (2001)
[2] O. Takeuchi and S. Akira. Pattern Recognition Receptors and Inflam(mation. Cell (2010)
[3] L.B. Ivashkiv and L.T. Donlin. Regulation of type I interferon responses. Nature Reviews Immunology (2013)
[4] J. Wu and Z.J. Cheng. Innate Immune Sensing and Signaling of Cytosolic Nucleic Acids. Annual Review of Immunology (2014)
[5] X. Cai et al. The cGAS-cGAMP-STING Pathway of Cytosolic DNA Sensing and Signaling. Cell (2014)
[6] D.J. Connolly and A.G. Bowie. The emerging role of human PYHIN proteins in innate immunity: implications for health and disease. Biochem Pharmacol. (2014)
[7] B.A. Diner et al. The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA. Mol Syst Biol. (2015)