The evolutionary landscape of cytosolic microbial sensors in humans

Host-pathogen interactions are generally initiated by host recognition of microbial components or danger signals triggered by microbial invasion. This recognition involves germline-encoded microbial sensors or pattern-recognition receptors (PRRs). By studying the way in which natural selection has driven the evolution of these microbial sensors in humans, we can identify genes playing an essential role and distinguish them from other, more redundant genes. We characterized the sequence diversity of the NOD-like receptor family, including the NALP and NOD/IPAF subfamilies, in various populations worldwide and compared this diversity with that of other PRR families, such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). We found that most NALPs had evolved under strong selective constraints, suggesting that their functions are essential and possibly much broader than previously thought. Conversely, most NOD/IPAF subfamily members were subject to more relaxed selective constraints, suggesting greater redundancy. Furthermore, some NALP genes, including NLRP1, NLRP14, and CIITA, were found to have evolved adaptively. We identified those variants conferring a selective advantage on some human populations as the most likely targets of positive selection. More generally, the strength of selection differed considerably between the major families of microbial sensors. Endosomal TLRs and most NALPs were found to evolve under stronger purifying selection than most NOD/IPAF subfamily members and cell-surface TLRs and RLRs, suggesting some degree of redundancy in the signaling pathways triggered by these molecules. This study provides novel perspectives and experimentally testable hypotheses concerning the relative biological relevance of the various families of microbial sensors in humans.     

NODs: intracellular proteins involved in inflammation and apoptosis

NOD (nucleotide-binding oligomerization domain) proteins are members of a family that includes the apoptosis regulator APAF1 (apoptotic protease activating factor 1), mammalian NOD-LRR (leucine-rich repeat) proteins and plant disease-resistance gene products. Several NOD proteins have been implicated in the induction of nuclear factor-kappaB (NF-kappaB) activity and in the activation of caspases. Two members of the NOD family, NOD1 and NOD2, mediate the recognition of specific bacterial components. Notably, genetic variation in the genes encoding the NOD proteins NOD2, cryopyrin and CIITA (MHC class II transactivator) in humans and Naip5 (neuronal apoptosis inhibitory protein 5) in mice is associated with inflammatory disease or increased susceptibility to bacterial infections. Mammalian NOD proteins seem to function as cytosolic sensors for the induction of apoptosis, as well as for innate recognition of microorganisms and regulation of inflammatory responses.     

Thin Filament Proteins and Thin Filament-Linked Regulation of Vertebrate Muscle Contractio

Abstract

The major intrinsic protein (MIP) of the bovine lens fiber cell membrane was the first member of the MIP family of proteins to be sequenced and characterized. It is probably a homotetramer with transmembrane channel activity that plays a role in lens biogenesis or maintenance. The polypeptide chain of each subunit may span the membrane six times, and both the N- and C-termini face the cell cytoplasm. Eighteen sequenced or partially sequenced proteins from bacteria, yeast, plants, and animals have now been shown to be members of the MIP family. These proteins appear to function in (1) metazoan development and neurogenesis (MIP and BIB), (2) water transport across the human erythrocyte membrane (ChIP), (3) communication between host plant cells and symbiotic nitrogen-fixing bacteria (NOD), (4) transport across the tonoplast membrane during plant seed development (α-TIP), (5) water stress-induced resistance to desiccation in plants (Wsi-TIP), (6) suppression of a genetic growth defect on fermentable sugars in yeast (FPS1), and (7) transport of glycerol across bacterial cell membranes (GlpF). One other sequenced member of the MIP family (ORF1 of Lactococcus lactis) has no known physiological function. The biochemical functions of the eukaryotic proteins are not well established.

Computer analyses have revealed that the first and second halves of all MTP family proteins probably arose by a tandem, intragenic, duplication event. Thus, the primary structure of putative transmembrane helices 1 to 3 is similar to that of putative transmembrane helices 4 to 6 even though they are of opposite orientation in the membrane. Among the most conserved residues in these two repeated halves are a membrane-embedded glutamate (E) in helices 1 and 4, an asparagine-proline-alanine (NPA) sequence in the loops between helices 2 and 3 (cytoplasmically localized) and helices 5 and 6 (extracellularly localized), and a glycine within helices 3 and 6. Statistical analyses suggest that the two halves of these proteins have evolved to serve distinct functions: the first half is more important for the generalized or common functions of these proteins, while the second half of these proteins is more differentiated to provide specific or dissimilar functions of the proteins. The apparent origin of MIP family proteins by duplication of a three-spanner precursor protein suggests an evolutionary origin distinct from other transport proteins with six transmembrane spanners. Based on the phylogenetic tree for the 18 sequenced members of the MTP family, we propose that a single, primordial gene arose in prokaryotes shortly before the emergence of eukaryotes, mat this gene was vertically transmitted to the principal eukaryotic kingdoms, and that subsequent gene duplication and divergence events gave rise to kingdom-related subfamilies or clusters of the MIP family.     

NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations.     

A new subfamily of major intrinsic proteins in plants

The major intrinsic proteins (MIPs) form a large protein family of ancient origin and are found in bacteria, fungi, animals, and plants. MIPs act as channels in membranes to facilitate passive transport across the membrane. Some MIPs allow small polar molecules like glycerol or urea to pass through the membrane. However, the majority of MIPs are thought to be aquaporins (AQPs), i.e., they are specific for water transport. Plant MIPs can be subdivided into the plasma membrane intrinsic protein, tonoplast intrinsic protein, and NOD26-like intrinsic protein subfamilies. By database mining and phylogenetic analyses, we have identified a new subfamily in plants, the Small basic Intrinsic Proteins (SIPs). Comparisons of sequences from the new subfamily with conserved amino acid residues in other MIPs reveal characteristic features of SIPs. Possible functional consequences of these features are discussed in relation to the recently solved structures of AQP1 and GlpF. We suggest that substitutions at conserved and structurally important positions imply a different substrate specificity for the new subfamily.     

Inflammasome components NALP 1 and 3 show distinct but separate expression profiles in human tissues suggesting a site-specific role in the inflammatory response.

Several autoinflammatory disorders such as Muckle-Wells syndrome are characterized by mutations in the NALP3/cryopyrin gene. NALP3 and NALP1 proteins can assemble to inflammasomes that activate caspase-1, resulting in the processing of pro-inflammatory cytokines IL-1beta and IL-18. The present study was designed to determine which cells and tissues express NALP1 and NALP3. Monoclonal antibodies were developed and their use revealed distinct distribution profiles of NALP1 and NALP3. Granulocytes, monocytes (very weakly), dendritic cells, and B and T cells all express NALP1 and NALP3. Highest levels of NALP1 are found in T cells and Langerhans cells. Furthermore, NALP1 is present in glandular epithelial structures such as stomach, gut, lung, and, surprisingly, in neurons and testis. In contrast to NALP1, NALP3 shows a more restricted tissue distribution with expression mainly in non-keratinizing epithelia in the oropharynx, esophagus, and ectocervix. Moreover, NALP3 expression is found in the urothelial layer in the bladder. Likewise, a difference in subcellular distribution between NALP1 and NALP3 is observed because NALP1 is localized mainly in the nucleus, whereas NALP3 is predominantly cytoplasmic. We propose that the presence of NALP3 in epithelial cells lining the oral and genital tracts allows the rapid sensing of invading pathogens, thereby triggering an innate immune response.     

Cutting edge: CATERPILLER: a large family of mammalian genes containing CARD,pyrin, nucleotide-binding,and leucine-rich repeat domains

Large mammalian proteins containing a nucleotide-binding domain (NBD) and C-terminal leucine-rich repeats (LRR) similar in structure to plant disease resistance proteins have been suggested as critical in innate immunity. Our interest in CIITA, a NBD/LRR protein, and recent reports linking mutations in two other NBD/LRR proteins to inflammatory disorders have prompted us to perform a search for other members. Twenty-two known and novel NBD/LRR genes are spread across eight human chromosomes, with multigene clusters occurring on 11, 16, and 19. Most of these are telomeric. Their N termini vary, but most have a pyrin domain. The genomic organization demonstrates a high degree of conservation of the NBD- and LRR-encoding exons. Except for CIITA, all the predicted NBD/LRR proteins are likely ATP-binding proteins. Some have broad tissue expression, whereas others are restricted to myeloid cells. The implications of these data on origins, expression, and function of these genes are discussed.     

Genome Wide Identification of C2H2-Type Zinc Finger Proteins of Tomato and Expression Analysis Under Different Abiotic Stresses

In plants, C2H2-type zinc finger proteins play important roles in multiple processes, including plant growth and development, as well as biotic and abiotic responses. In the present study, based on the presence of the C2H2 domain (CX2~4CX3FX5LX2HX3~5H), 112 C2H2-type zinc finger proteins were predicted in tomato. Through gene and protein structures analyses and phylogenetic analysis, the 112 C2H2-type zinc finger proteins were divided into five subfamilies. Members of the same subfamily shared similarities in gene and protein structures, while members of different subfamilies contained different numbers of the C2H2 domain. The tissue expression pattern analysis showed that 24 C2H2-type zinc finger proteins are constitutively expressed in all tissues, indicating that they may play important roles in the growth and development of all tissues. In addition, under chilling (4 °C), heat (42 °C), high salinity (200 Mm NaCl), and osmotic (20% PEG) stresses, members of C2H2-type zinc finger family were induced to varying degrees, which suggested that these genes were involved in multiple abiotic stress responses. This study will provide theoretical basis for further research of C2H2-type zinc finger proteins in tomato.

     

Evolutionary diversification of the avian fatty acid-binding proteins

Phylogenetic analysis of avian and other vertebrate fatty acid binding proteins (FABPs) supported the hypothesis that several gene duplications within this family occurred prior to the most recent common ancestor (MRCA) of tetrapods and bony fishes. The chicken genome encodes two liver-expressed FABPs: (1) L-FABP or FABP1; and (2) Lb-FABP. We propose that the latter be designated FABP10, because in our phylogenetic analysis it clustered with zebrafish FABP10. Bioinformatic analysis of across-tissue gene expression patterns in the chicken showed some congruence with phylogenetic relationships. On the basis of expression, chicken FABP genes seemed to form two major groups: (1) a cluster of genes many of which showed predominant expression in the digestive system (FABP1, FABP2, FABP6, FABP10, RBP1, and CRABP1); and (2) a cluster of genes most of which had predominant expression in tissues other than those of the digestive system, including muscle and the central nervous system (FABP3, FABP4, FABP5, FABP7, and PMP2). Since these clusters corresponded to major clusters in the phylogenetic tree as well, it seems a plausible hypothesis that the earliest duplication in the vertebrate FABP family led to the divergence of a gut-specialized gene from a gene expressed mainly in nervous and muscular systems. Data on gene expression in livers of two lines of chickens selected for high growth and low growth showed differences between FABP1 and FABP10 expressions in the liver, supporting the hypothesis of functional divergence between the two chicken liver-expressed FABPs related to food intake.     

The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants

Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species.     

Self-association of CIITA and its transactivation potential.

The major histocompatibility complex (MHC) class II transactivator (CIITA) regulates the expression of genes involved in the immune response, including MHC class II genes and the interleukin-4 gene. Interactions between CIITA and sequence-specific, DNA-binding proteins are required for CIITA to function as an activator of MHC class II genes. CIITA also interacts with the coactivators CBP (also called p300), and this interaction leads to synergistic activation of MHC class II promoters. Here, we report that CIITA forms complexes with itself and that a central region, including the GTP-binding domain is sufficient for self-association. Additionally, this central region interacts with the C-terminal leucine-rich repeat as well as the N-terminal acidic domain. LXXLL motifs residing in the GTP-binding domain are essential for self-association. Finally, distinct differences exist among various CIITA mutant proteins with regard to activation function, subcellular localization, and association with wild-type protein and dominant-negative potential.     

Origin and diversification of the L-amino oxidase family in innate immune defenses of animals

L-amino acid oxidases (LAOs), because they produce hydrogen peroxide as a by-product, function in innate immune defenses of both vertebrates and mollusks. Phylogenetic analysis revealed two major subfamilies of LAOs: (1) a subfamily including LAOs from vertebrates and mainly from Terrabacteria and (2) a subfamily including LAOs from mollusks and Hydrobacteria. These subfamilies thus originated early in the history of life, implying that their innate immune functions in vertebrates and mollusks have evolved separately. Mammalian LAOs were found to belong to three separate clades: (1) LAO1, (2) LAO2, and (3) IL4I1. Phylogenetic analysis supported the hypothesis that LAO1 and LAO2 arose by a gene duplication prior to the divergence of marsupials from placental mammals, while IL4I1 duplicated from the ancestor of the LAO1 and LAO2 prior to the divergence of tetrapods from bony fishes. Mammalian IL4I1 clustered with LAOs from bony fishes, and these molecules shared a number of unique sequence features, including both amino acid replacements and a unique two-codon deletion. It is certain such unique features may be functionally important, especially three unique amino acid replacements in close proximity to the putative active site.