Regulation of Immunity by Butyrophilins.

Butyrophilin molecules (commonly contracted to BTN), collectively take their name from the eponymous protein in cow's milk. They are considered to be members of the B7 family of costimulatory receptors, which includes B7.1 (CD80), B7.2 (CD86), and related molecules, such as PD-L1 (B7-H1, CD274), ICOS-L (CD275), and B7-H3 (CD276). These coreceptors modulate T cell responses upon antigen presentation by major histocompatibility complex and cognate αß T cell receptor engagement. Molecules such as BTN3A1 (CD277), myelin oligodendrocyte glycoprotein, and mouse Skint1 and Btnl2, all members of the butyrophilin family, show greater structural and functional diversity than the canonical B7 receptors. Some butyrophilins mediate complex interactions between antigen-presenting cells and conventional αß T cells, and others regulate the immune responses of specific γδ T cell subsets by mechanisms that have characteristics of both innate and adaptive immunity.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1.

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel ß-sheets of seven and six strands, arranged as a distorted ß-sandwich. Furthermore, two long loops partially cover the concave face of the ß-sandwich defined by the ß-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Innate immune genes of the chicken MHC and related regions.

Compared to the major histocompatibility complex (MHC) of typical mammals, the chicken BF/BL region is small and simple, with most of the genes playing central roles in the adaptive immune response. However, some genes of the chicken MHC are almost certainly involved in innate immunity, such as the complement component C4 and the lectin-like receptor/ligand gene pair BNK and Blec. The poorly expressed classical class I molecule BF1 is known to be recognised by natural killer (NK) cells and, analogous to mammalian immune responses, the classical class I molecules BF1 and BF2, the CD1 homologs and the butyrophilin homologs called BG may be recognised by adaptive immune lymphocytes with semi-invariant receptors in a so-called adaptate manner. Moreover, the TRIM and BG regions next to the chicken MHC, along with the genetically unlinked Y and olfactory/scavenger receptor regions on the same chromosome, have multigene families almost certainly involved in innate and adaptate responses. On this chicken microchromosome, the simplicity of the adaptive immune gene systems contrasts with the complexity of the gene systems potentially involved in innate immunity.

Recurrent expansions of B30.2-associated immune receptor families in fish.

B30.2 domains, also known as PRY/SPRY, are key components of specific subsets of two large families of proteins involved in innate immunity: the tripartite motif proteins (TRIMs) and the Nod-like receptors (NLRs). TRIM proteins are important, often inducible factors of antiviral innate immunity, targeting multiple steps of viral cycles through a variety of mechanisms. NLRs prime and regulate systemic innate defenses, especially against bacteria, and control inflammation. Large TRIM and NLR subsets characterized by the presence of a B30.2 domain have been reported from a few fish species including zebrafish and seem to be strongly prone to gene duplication/expansion. Here, we performed a large-scale survey of these receptors across about 150 fish genomes, focusing on ray-finned fishes. We assessed the number and genomic distribution of domains and domain combinations associated with TRIMs, NLRs, and other genes containing B30.2 domains and looked for gene expansion patterns across fish groups. We then used a model to test the impact of taxonomy, genome size, and environmental variables on the copy numbers of these genes. Our findings reveal novel domain structures, clade-specific gains and losses. They also assist with the timing of the gene expansions, reveal patterns associated with the MHC, and lay the groundwork for further studies delving deeper into the forces that drive the copy number variation of immune genes on a species level.

Crystal structure of the SPRY domain-containing protein 7 reveals unique structural features.

The SPRY/B30.2 domain is one of the most abundant protein domains found in eukaryotes. Vast majority of the SPRY domain-containing proteins are multi-domain proteins. The SPRY domain-containing protein 7 (SPRY7, also named C13orf1, and named chronic lymphocytic leukemia deletion region gene 6 protein, CCLD6, encoded by the spryd7 gene) is the smallest SPRY domain protein in human that does not contain other accessory domains. Here we have determined the crystal structure of human SPRY7 at a resolution of 1.62 Å and found that SPRY7 has some unique structural features that are not present in other previously reported SRPY domain structures. Overall, SPRY7 may represent an evolutionary early version of the SPRY domain, and subsequent loop insertions and expansions, residue substitutions, as well as domain combinations have rendered the SPRY domain versatile binding specificities and broad biological functions. These results serve as a useful basis for a profound characterization of the molecular interactions of SPRY7.

The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α.

Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.

Identification of a second binding site on the TRIM25 B30.2 domain.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

A Photo-Crosslinkable Biotin Derivative of the Phosphoantigen (E)-4-Hydroxy-3-Methylbut-2-Enyl Diphosphate (HMBPP) Activates Vγ9Vδ2 T Cells and Binds to the HMBPP Site of BTN3A1.

Vγ9Vδ2 T cells play an important role in the cross talk of the innate and adaptive immune system. For their activation by phosphoantigens (PAgs), both cell surface receptors, the eponymous Vγ9Vδ2 T cell antigen receptors (Vγ9Vδ2 TCRs) on Vγ9Vδ2 T cells and butyrophilin 3A1 (BTN3A1) on the phosphoantigen-"presenting" cell, are mandatory. To find yet undetected but further contributing proteins, a biotinylated, photo-crosslinkable benzophenone probe BioBP-HMBPP (2) was synthesized from a known allyl alcohol in nine steps and overall 16 % yield. 2 is based on the picomolar PAg (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1). Laser irradiation of 2 at 308 nm initiated the photo-crosslinking reaction with proteins. When the B30.2 domain of BTN3A1, which contains a positively charged PAg-binding pocket, was exposed to increasing amounts of HMBPP (1), labeling by BioBP-HMBPP (2) was reduced significantly. Because BSA labeling was not impaired, 2 clearly binds to the same site as natural ligand 1. Thus, BioBP-HMBPP (2) is a suitable tool to identify co-ligands or receptors involved in PAg-mediated T cell activation.

Human gamma delta T cells: Evolution and ligand recognition.

The γδ T cell lineage in humans remains much of an enigma due to the low number of defined antigens, the non-canonical ways in which these cells respond to their environment and difficulty in tracking this population in vivo. In this review, we survey a comparative evolutionary analysis of the primate V, D and J gene segments and contrast these findings with recent progress in defining antigen recognition by different populations of γδ T cells in humans. Signatures of both purifying and diversifying selection at the Vδ and Vγ gene loci are placed into context of Vδ1+ γδ T cell recognition of CD1d presenting different lipids, and Vγ 9Vδ2 T cell modulation by pyrophosphate-based phosphoantigens through the butyrophilins BTN3A. From this comparison, it is clear that co-evolution between γδ TCRs and these ligands is likely occurring, but the diversity inherent in these recombined receptors is an important feature in ligand surveillance.

[Influence of M680I and M694V mutations on pyrin's domain B30.2 tertiary structure and it's complex formation ability with caspase-1].

The M680I and M694V mutations located in the B30.2 pyrin domain are responsible for the manifestation of the most common forms of Familial Mediterranean fever. It is well known that a malfunction of the pyrin-caspase-1 complex is the main cause of inflammation in FMF. The purpose of this study was to identify possible changes in the tertiary structure of mutated B30.2 domain and to determine their potential consequences in the formation of the pyrin-caspase-1 complex. Using computer modeling, it was found that the above mutations change the tertiary structure of B30.2 domain, causing shifts of binding sites and altering the energy of interaction between B30.2 and caspase-1.

TRIM103 activates the RLRs pathway to enhance antiviral response by targeting VP5 and VP7.

Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.

Proteomic analysis of the NOS2 interactome in human airway epithelial cells.

The cytokine-inducible isoform of nitric oxide synthase (NOS2) is constitutively expressed in human respiratory epithelia and is upregulated in inflammatory lung disease. Here, we sought to better define the protein interactions that may be important for NOS2 activity and stability, as well as to identify potential targets of NOS2-derived NO, in the respiratory epithelium. We overexpressed Flag-tagged, catalytically-inactive NOS2 in A549 cells and used mass spectrometry to qualitatively identify NOS2 co-immunoprecipitating proteins. Stable isotope labeling of amino acids in cell culture (SILAC) was used to quantify the coordinate effects of cytokine stimulation on NOS2-protein interactions. Multi-protein networks dominated the NOS2 interactome, and cytokine-inducible interactions with allosteric activators and with the ubiquitin-proteasome system were correlated with cytokine-dependent increases in NO metabolites and in NOS2 ubiquitination. The ubiquitin ligase scaffolding protein, FBXO45, was identified as a novel, direct NOS2 interactor. Similar to the SPRY domain-containing SOCS box (SPSB) proteins, FBXO45 requires Asn27 in the (23)DINNN(27) motif of NOS2 for its interaction. However, FBXO45 is unique from the SPSBs in that it recruits a distinct E3 ligase complex containing MYCBP2 and SKP1. Collectively, these findings demonstrate the general utility of interaction proteomics for defining new aspects of NOS2 physiology.

Evolution and expression of the duck TRIM gene repertoire.

Tripartite motif (TRIM) proteins are involved in development, innate immunity, and viral restriction. TRIM gene repertoires vary between species, likely due to diversification caused by selective pressures from pathogens; however, this has not been explored in birds. We mined a de novo assembled transcriptome for the TRIM gene repertoire of the domestic mallard duck (Anas platyrhynchos), a reservoir host of influenza A viruses. We found 57 TRIM genes in the duck, which represent all 12 subfamilies based on their C-terminal domains. Members of the C-IV subfamily with C-terminal PRY-SPRY domains are known to augment immune responses in mammals. We compared C-IV TRIM proteins between reptiles, birds, and mammals and show that many C-IV subfamily members have arisen independently in these lineages. A comparison of the MHC-linked C-IV TRIM genes reveals expansions in birds and reptiles. The TRIM25 locus with related innate receptor modifiers is adjacent to the MHC in reptile and marsupial genomes, suggesting the ancestral organization. Within the avian lineage, both the MHC and TRIM25 loci have undergone significant TRIM gene reorganizations and divergence, both hallmarks of pathogen-driven selection. To assess the expression of TRIM genes, we aligned RNA-seq reads from duck tissues. C-IV TRIMs had high relative expression in immune relevant sites such as the lung, spleen, kidney, and intestine, and low expression in immune privileged sites such as in the brain or gonads. Gene loss and gain in the evolution of the TRIM repertoire in birds suggests candidate immune genes and potential targets of viral subversion.

Dynamic disequilibrium-based pathogenicity model in mutated pyrin's B30.2 domain-Casp1/p20 complex.

BACKGROUND: The B30.2 variants lead to most relevant severity forms of familial Mediterranean fever (FMF) manifestations. The B30.2 domain plays a key role in protein-protein interaction (PPI) of pyrin with other apoptosis proteins and in regulation the cascade of inflammatory reactions. Pyrin-casp1 interaction is mainly responsible for the dysregulation of the inflammatory responses in FMF. Lower binding affinity was observed between the mutant B30.2 pyrin and casp1 without the release of the complete pathogenicity mechanism. The aim of this study was to identify the possible effects of the interface pocked residues in B30.2/SPRY-Casp1/p20 complex using molecular mechanics simulation and in silico analysis. RESULTS: It was found that Lys671Met, Ser703Ile, and Ala744Ser variants led mainly to shift of the binding affinity (∆G), dissociation constant (Kd), and root mean square deviation (RMSD) in B30.2/SPRY-Casp1/p20 complex leading to dynamic disequilibrium of the p20-B30.2/SPRY complex toward its complex form. The current pathogenicity model and its predicted implementation in the relevant colchicine dosage were delineated. CONCLUSION: The molecular mechanics analysis of B30.2/SPRY-p20 complex harboring Lys671Met, Ser703Ile, and Ala744Ser variants showed dynamic disequilibrium of B30.2/SPRY-casp1/p20complex in context of the studied variants that could be a new computational model for FMF pathogenicity. This study also highlighted the specific biochemical markers that could be useful to adjust the colchicine dose in FMF patients.

Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism.

The pyrin inflammasome acts as a guard of RhoA GTPases and is central to immune defenses against RhoA-manipulating pathogens. Pyrin activation proceeds in two steps. Yet, the second step is still poorly understood. Using cells constitutively activated for the pyrin step 1, a chemical screen identifies etiocholanolone and pregnanolone, two catabolites of testosterone and progesterone, acting at low concentrations as specific step 2 activators. High concentrations of these metabolites fully and rapidly activate pyrin, in a human specific, B30.2 domain-dependent manner and without inhibiting RhoA. Mutations in MEFV, encoding pyrin, cause two distinct autoinflammatory diseases pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). Monocytes from PAAND patients, and to a lower extent from FMF patients, display increased responses to these metabolites. This study identifies an unconventional pyrin activation mechanism, indicates that endogenous steroid catabolites can drive autoinflammation, through the pyrin inflammasome, and explains the "steroid fever" described in the late 1950s upon steroid injection in humans.

Molecular characterization and expression pattern of tripartite motif protein 39 in Gallus gallus with a complete PRY/SPRY domain.

Members of tripartite motif (TRIM) proteins in mammals play important roles in multiple cellular processes in the immune system. In the present study we have obtained the chicken TRIM39 with the insertion of a base A at position 1006 bp, compared to the sequence in the NCBI database (Accession No: NM 001006196), which made TRIM39 fulfill the TRIM rule of domain composition with both PRY, and SPRY domains. The open reading frame consisted of 1392 bp encoding 463 amino acid residues. The amino acid sequences of TRIM39 protein in mammals were highly similar (from 91.48% to 99.61%), while chicken TRIM39 had relatively low homology with mammals (from 29.2% to 39.59%). Real time RT-PCR indicated that the mRNA expression level of TRIM39 was the highest in spleen, with a lower expression in liver, brain, and lung, suggesting it might be an important protein participating in the immune system.

Genome-Wide Identification and Expression Analysis of Potential Antiviral Tripartite Motif Proteins (TRIMs) in Grass Carp (Ctenopharyngodon idella).

Tripartite motif proteins (TRIMs), especially B30.2 domain-containing TRIMs (TRIMs-B30.2), are increasingly well known for their antiviral immune functions in mammals, while antiviral TRIMs are far from being identified in teleosts. In the present study, we identified a total of 42 CiTRIMs from the genome of grass carp, Ctenopharyngodon idella, an important cultured teleost in China, based on hmmsearch and SMART analysis. Among these CiTRIMs, the gene loci of 37 CiTRIMs were located on different chromosomes and shared gene collinearities with homologous counterparts from human and zebrafish genomes. They possessed intact conserved RBCC or RB domain assemblies at their N-termini and eight different domains, including the B30.2 domain, at their C-termini. A total of 19 TRIMs-B30.2 were identified, and most of them were clustered into a large branch of CiTRIMs in the dendrogram. Tissue expression analysis showed that 42 CiTRIMs were universally expressed in various grass carp tissues. A total of 11 significantly differentially expressed CiTRIMs were found in two sets of grass carp transcriptomes during grass carp reovirus (GCRV) infection. Three of them, including Cibtr40, CiTRIM103 and CiTRIM109, which all belonged to TRIMs-B30.2, were associated with the type I interferon response during GCRV infection by weighted network co-expression and gene expression trend analyses, suggesting their involvement in antiviral immunity. These findings may offer useful information for understanding the structure, evolution, and function of TRIMs in teleosts and provide potential antiviral immune molecule markers for grass carp.

Crystal structure of the SPRY domain of human SPSB2 in the apo state.

The SPRY domain-containing SOCS box protein 2 (SPSB2) is one of four mammalian SPSB proteins that are characterized by a C-terminal SOCS box and a central SPRY/B30.2 domain. SPSB2 interacts with inducible nitric oxide synthase (iNOS) via the SPRY domain and polyubiquitinates iNOS, resulting in its proteasomal degradation. Inhibitors that can disrupt SPSB2-iNOS interaction and augment NO production may serve as novel anti-infective and anticancer agents. The previously determined murine SPSB2 structure may not reflect the true apo conformation of the iNOS-binding site. Here, the crystal structure of human SPSB2 SPRY domain in the apo state is reported at a resolution of 1.9 Å. Comparison of the apo and ligand-bound structures reveals that the iNOS-binding site is highly preformed and that major conformational changes do not occur upon ligand binding. Moreover, the C-terminal His6 tag of the recombinant protein binds to a shallow pocket adjacent to the iNOS-binding site on a crystallographically related SPSB2 molecule. These findings may help in structure-based and fragment-based SPSB2 inhibitor design in the future.

Towards Deciphering the Hidden Mechanisms That Contribute to the Antigenic Activation Process of Human Vγ9Vδ2 T Cells.

Vγ9Vδ2 T cells represent a major unconventional γδ T cell subset located in the peripheral blood of adults in humans and several non-human primates. Lymphocytes that constitute this transitional subset can sense subtle level changes of intracellular phosphorylated intermediates of the isoprenoid biosynthesis pathway (phosphoantigens, pAg), such as isopentenyl pyrophosphate, during cell stress events. This unique antigenic activation process operates in a rigorous framework that requires the expression of butyrophilin 3A1 (BTN3A1/CD277) molecules, which are type I glycoproteins that belong to the B7 family. Several studies have further shown that pAg specifically bind to the intracellular B30.2 domain of BTN3A1 linked to the antigenic activation of Vγ9Vδ2 T cells. Here, we highlight the recent advances in BTN3A1 dynamics induced upon the binding of pAg and the contribution of the different subunits to this activation process. Recent reports support that conformational modifications of BTN3A1 might represent a key step in the detection of infection or tumorigenesis by Vγ9Vδ2 T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that target defined functions of this unique γδ T cell subset.

Targeting TRIM5α in HIV Cure Strategies for the CRISPR-Cas9 Era.

In the past decade, studies of innate immune activity against HIV-1 and other retroviruses have revealed a powerful array of host factors that can attack the virus at various stages of its life cycle in human and primate cells, raising the prospect that these antiviral factors could be manipulated in immunotherapeutic strategies for HIV infection. This has not proved straightforward: while HIV accessory genes encode proteins that subvert or destroy many of these restriction factors, others, such as human TRIM5α show limited potency against HIV-1. However, HIV-1 is much more susceptible to simian versions of TRIM5α: could this information be translated into the development of an effective gene therapy for HIV infection? Reigniting research into the restriction factor TRIM5α in the era of superior gene editing technology such as CRISPR-Cas9 presents an exciting opportunity to revisit this prospect.