Structural localization of disease-associated sequence variations in the NACHT and LRR domains of PYPAF1 and NOD2

Several autoinflammatory diseases with distinct clinical manifestations have been associated with sequence variations in the gene products PYPAF1/CIAS1 and NOD2/CARD15. Both proteins belong to the PYD/CARD-containing family of apoptosis regulators and activators of pro-inflammatory caspases. To gain insight into the dysfunctional role of sequence alterations, we assembled a structure-based multiple sequence alignment of family members and related proteins. This allowed us to analyze the putative effect of the alterations on the function of nucleotide-binding (NACHT) and leucine-rich repeat (LRR) domains shared by the family members. In support of this analysis, we carefully selected template structures for the NACHT and LRR domains and mapped the genetic variations onto 3D domain models. Additionally, we propose a model of the NACHT and LRR domain complex. Our study revealed that many of the disease-associated sequence variants are located close to highly conserved sequence regions of functional relevance and are spatially adjacent in the predicted 3D structure. The implications on the domain functions such as NTP-hydrolysis or oligomerization are discussed.     

Identification of mammalian orthologs associates PYPAF5 with distinct functional roles

PYRIN- and CARD-containing proteins belong to a recently identified protein family involved in the regulation of apoptosis and inflammatory processes. Variations in the gene products of the family members PYPAF1 and NOD2/CARD15 have been associated with several autoinflammatory diseases. We could identify the mouse orthologs of PYPAF1, PYPAF5, NOD1, NOD2 and the rat ortholog of PYPAF5. Intriguingly, we found that PYPAF5 has been reported previously not only as regulator of NF-kappaB and caspase-1, but also as angiotensin II and vasopressin receptor. In particular, based on a comprehensive sequence analysis, we propose a structural model for this hormone receptor that is different from the model suggested previously.     

Monarch-1/PYPAF7 and other CATERPILLER (CLR, NOD, NLR) proteins with negative regulatory functions

CATERPILLER is a mammalian gene family with signature NBD and LRR domains. Several members of this family are positive regulators of inflammatory responses. Others, however, exert negative effects on proinflammatory responses. These data are particularly convincing when shRNA/siRNA are used. This review focuses on the Monarch-1/PYPAF7 gene with brief discussions of CLR16.2/NOD3, PYPAF2/PAN1/NALP2, and PYPAF3.     

Mutational analysis of human NOD1 and NOD2 NACHT domains reveals different modes of activation

Nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are intracellular pattern recognition receptors (PRRs) of the nucleotide-binding domain and leucine-rich repeat containing (NLR) gene family involved in innate immune responses. Their centrally located NACHT domain displays ATPase activity and is necessary for activation and oligomerization leading to inflammatory signaling responses. Mutations affecting key residues of the ATPase domain of NOD2 are linked to severe auto-inflammatory diseases, such as Blau syndrome and early-onset sarcoidosis. By mutational dissection of the ATPase domain function, we show that the NLR-specific extended Walker B box (DGhDE) can functionally replace the canonical Walker B sequence (DDhWD) found in other ATPases. A requirement for an intact Walker A box and the magnesium-co-ordinating aspartate of the classical Walker B box suggest that an initial ATP hydrolysis step is necessary for activation of both NOD1 and NOD2. In contrast, a Blau-syndrome associated mutation located in the extended Walker B box of NOD2 that results in higher autoactivation and ligand-induced signaling does not affect NOD1 function. Moreover, mutation of a conserved histidine in the NACHT domain also has contrasting effects on NOD1 and NOD2 mediated NF-κB activation. We conclude that these two NLRs employ different modes of activation and propose distinct models for activation of NOD1 and NOD2.     

PYPAF1, a PYRIN-containing Apaf1-like protein that assembles with ASC and regulates activation of NF-kappa B.

The PYRIN domain is a recently identified protein-protein interaction domain that is found at the N terminus of several proteins thought to function in apoptotic and inflammatory signaling pathways. We report here that PYPAF1 (PYRIN-containing Apaf1-like protein 1) is a novel PYRIN-containing signaling protein that belongs to the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signaling proteins. The expression of PYPAF1 is highly restricted to immune cells, and its gene maps to chromosome 1q44, a locus that is associated with the rare inflammatory diseases Muckle-Wells syndrome and familial cold urticaria. To identify downstream signaling partners of PYPAF1, we performed a mammalian two-hybrid screen and identified ASC as a PYRIN-containing protein that interacts selectively with the PYRIN domain of PYPAF1. When expressed in cells, ASC recruits PYPAF1 to distinct cytoplasmic loci and induces the activation of NF-kappaB. Furthermore, coexpression of PYPAF1 with ASC results in a potent synergistic activation of NF-kappaB. These findings suggest that PYPAF1 and ASC function as upstream activators of NF-kappaB signaling.     

Genetic polymorphisms and haplotype structures of the CYP4A22 gene in a Japanese population

The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.     

番茄MADS-box基因Lemads1的可变剪接与表达模式分析

可变剪接(alternative splicing) 是真核基因转录的普遍现象和重要调控方式,在生物体的各种生理过程中发挥着重要的作用。植物转录因子MADS box基因在植物生长发育中发挥重要作用。番茄(Solanum lycopersicum L.) SEPALLATA (SEP) 亚族基因Lemads1是1个在番茄果实成熟过程中具有关键作用的MADS box家族成员,但至今尚未见其转录剪接模式的详细报道。本研究发现,Lemads1基因在转录时存在可变剪接现象,并检测到4种Lemads1可变剪接转录本,其扩增长度依次为：699 bp、741 bp、1 404 bp和1 539 bp。剪接方式有外显子选择性切除和内含子保留两种。这4种转录本编码长度为232 aa、246 aa和266 aa的3种蛋白质。氨基酸序列比对和系统进化分析结果表明,这3种蛋白序列的差异主要集中在C区,但并不影响它们的系统进化地位。在进化上,它们仍属于SEP亚家族LOFSEP亚支中的FBP9/23分支,与它们亲缘关系最近的番茄SEP类蛋白是SLMBP21。Lemads1反义RNA转基因植株表型观察结果表明,该基因可能参与番茄果实成熟和萼片发育过程;而组织特异性表达分析表明,该基因的4种转录本具有相似但并不完全相同的表达模式,它们都在萼片和果实中高表达,但在这2个组织发育过程中的表达变化却有明显差异。暗示这些可变剪接转录本在萼片和果实发育过程中扮演了不同的角色。本研究提供了番茄Lemads1基因存在可变剪切的重要信息,对该基因功能的深度发掘具有指导意义。     

Non-association of Crohn's disease with NOD2 gene variants in Moroccan patients

Crohn's disease is a chronic inflammatory bowel disease, with multifactorial traits, that can involve any part of the gastrointestinal tract. In recent years, a dozen genome-wide association scan and meta-analysis were published bringing the number of susceptibility alleles to more than 30 variations. However, the major susceptibility gene for Crohn's disease is NOD2, located on proximal 16q, which is involved in the innate immune response. Three main variants of this gene: two single nucleotide polymorphisms p.Arg702Trp and p.Gly908Arg substitutions and frameshift polymorphism p.Leu1007fsinsC are involved in susceptibility to Crohn's disease.     

PYPAF3, a PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1-dependent interleukin-1beta secretion

PYPAF3 is a member of the PYRIN-containing apoptotic protease-activating factor-1-like proteins (PYPAFs, also called NALPs). Among the members of this family, PYPAF1, PYPAF5, PYPAF7, and NALP1 have been shown to induce caspase-1-dependent interleukin-1beta secretion and NF-kappaB activation in the presence of the adaptor molecule ASC. On the other hand, we recently discovered that PYNOD, another member of this family, is a suppressor of these responses. Here, we show that PYPAF3 is the second member that inhibits caspase-1-dependent interleukin-1beta secretion. In contrast, PYPAF2/NALP2 does not inhibit this response but rather inhibits the NF-kappaB activation that is induced by the combined expression of PYPAF1 and ASC. Both PYPAF2 and PYPAF3 mRNAs are broadly expressed in a variety of tissues; however, neither is expressed in skeletal muscle, and only PYPAF2 mRNA is expressed in heart and brain. They are also expressed in many cell lines of both hematopoietic and non-hematopoietic lineages. Stimulation of monocytic THP-1 cells with lipopolysaccharide or interleukin-1beta induced PYPAF3 mRNA expression. Furthermore, the stable expression of PYPAF3 in THP-1 cells abrogated the ability of the cells to produce interleukin-1beta in response to lipopolysaccharide. These results suggest that PYPAF3 is a feedback regulator of interleukin-1beta secretion. Thus, PYPAF2 and PYPAF3, together with PYNOD, constitute an anti-inflammatory subgroup of PYPAFs.     

PYPAF7, a novel PYRIN-containing Apaf1-like protein that regulates activation of NF-kappa B and caspase-1-dependent cytokine processing

PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-kappa B. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1 beta. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-kappa B and cytokine processing.     

Studies on structure-based sequence alignment and phylogenies of beta-lactamases

The β-lactamases enzymes cleave the amide bond in β-lactam ring, rendering β-lactam antibiotics harmless to bacteria. In this communication we have studied structure-function relationship and phylogenies of class A, B and D beta-lactamases using structure-based sequence alignment and phylip programs respectively. The data of structure-based sequence alignment suggests that in different isolates of TEM-1, mutations did not occur at or near sequence motifs. Since deletions are reported to be lethal to structure and function of enzyme. Therefore, in these variants antibiotic hydrolysis profile and specificity will be affected. The alignment data of class A enzyme SHV-1, CTX-M-15, class D enzyme, OXA-10, and class B enzyme VIM-2 and SIM-1 show sequence motifs along with other part of polypeptide are essentially conserved. These results imply that conformations of betalactamases are close to native state and possess normal hydrolytic activities towards beta-lactam antibiotics. However, class B enzyme such as IMP-1 and NDM-1 are less conserved than other class A and D studied here because mutation and deletions occurred at critically important region such as active site. Therefore, the structure of these beta-lactamases will be altered and antibiotic hydrolysis profile will be affected. Phylogenetic studies suggest that class A and D beta-lactamases including TOHO-1 and OXA-10 respectively evolved by horizontal gene transfer (HGT) whereas other member of class A such as TEM-1 evolved by gene duplication mechanism. Taken together, these studies justify structure-function relationship of beta-lactamases and phylogenetic studies suggest these enzymes evolved by different mechanisms.     

Induction of PYPAF1 during in vitro maturation of mouse mast cells

Coculture of mouse bone marrow-derived immature mast cells (BMMC) with Swiss 3T3 fibroblasts in the presence of stem cell factor (SCF) promotes morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype, which is accompanied by increased expression of several unique genes. Here we report the molecular identification of one of them, mast cell maturation-associated inducible gene (MMIG)-1. The MMIG-1 cDNA encodes a 117-kDa cytosolic protein that comprises an N-terminal PYRIN domain, a central nucleotide-binding domain, and nine C-terminal leucine-rich repeats. MMIG-1 shows >85% sequence similarity to human cryopyrin/PYPAF1, a causal gene for familial cold urticaria and Muckle-Wells syndrome. MMIG-1 was distributed in the cytosol of CTMC-like differentiated BMMC. MMIG-1 underwent alternative splicing in the leucine-rich repeats and each variant was induced differently in BMMC during coculture. Moreover, its expression was increased in the ears of mice with experimental atopic dermatitis. Thus, MMIG-1, a likely mouse PYPAF1 ortholog, may play a role in mast cell-directed inflammatory diseases.     

Sequence similarity between Photosystems I and II. Identification of a Photosystem I reaction center transmembrane helix that is similar to transmembrane helix IV of the D2 subunit of Photosystem II and the M subunit of the non-sulfur purple and flexible green bacteria

There are basic structural similarities between plant PS II and bacterial RCs of the Chloroflexaceae and Rhodospirillaceae. These RCs are referred to as PS II-type RCs. A similar relationship of PS I RC to PS II-type RCs has not been established. Although plant PS I and PS II RCs show structural and functional differences, they also share similarities. Therefore, the A and B polypeptides of PS I were searched for PS II D1 and D2 polypeptide-like sequences. An alignment without gaps was found between PS II-type D2/M helix IV and PS I B helix X, as well as a weaker alignment of PS II-type D1/L with PS I B helix X. No comparable alignment with PS I A was found. In the M/D2 alignment there were eight identities and some conservative substitutions in twenty nine residues. PS I B helix X appeared to contain a modified chlorophyll dimer and monomer binding site and a modified non-heme iron-quinone binding site. The conserved residue sequence was found only in RC polypeptides. The proposed chlorophyll dimer-monomer binding site was located transmembrane from the iron-sulfur cluster X binding site. The conserved residues generally are those that interact with prosthetic groups. Half of the conserved residues are located on the same side of the helix. Thus, although there are impediments to concluding firmly that PS I B helix X has a functional and evolutionary relatedness to the D2 PS II and bacterial M RC polypeptides, our analysis gives reasonable support to the idea.Abbreviation RC reaction center     

Extensive regions of homology in front of the two hsp70 heat shock variant genes in Drosophila melanogaster

The major heat shock protein of 70,000 M_r in Drosophila melanogaster is encoded by two variant gene types located, respectively, at the chromosomal sites 87A7 and 87C1. We present the DNA sequence of a complete hsp70² gene of the 87A7 type and of the adjacent regions from both variants, extending to 1·2 × 10³ bases upstream from the start of the messenger coding region. We find an untranslated region of 250 nucleotides at the 5′ end of the messenger coding sequence in both variants. There is only one open reading frame which allows coding of a 70,000 M_r protein within the 87A7 variant, as found for an 87C1 variant (Ingolia et al., 1980). We observe 4·2% nucleotide divergence between these two variants with complete conservation of the reading frame. There is a conserved sequence of 355 nucleotides in front of each hsp70 gene, which is 85% homologous between the two variants. The presence of the same sequence element in γ, in front of the αβ heat shock genes (R. W. Hackett & J. T. Lis, personal communication) suggests that this element contains the regulatory signals for the coordinate expression of both the hsp70 and the αβ heat shock genes. Finally we find a very A + T-rich sequence of 150 basepairs which is highly conserved (91·8%) 0·6 × 10³ bases upstream from two hps70 gene variants.     

Role of a SpoVA protein in dipicolinic acid uptake into developing spores of Bacillus subtilis

The proteins encoded by the spoVA operon, including SpoVAD, are essential for the uptake of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA(2,6)) and Ca(2+) into developing spores of the bacterium Bacillus subtilis. The crystal structure of B. subtilis SpoVAD has been determined recently, and a structural homology search revealed that SpoVAD shares significant structural similarity but not sequence homology to a group of enzymes that bind to and/or act on small aromatic molecules. We find that molecular docking placed DPA(2,6) exclusively in a highly conserved potential substrate-binding pocket in SpoVAD that is similar to that in the structurally homologous enzymes. We further demonstrate that SpoVAD binds both DPA(2,6) and Ca(2+)-DPA(2,6) with a similar affinity, while exhibiting markedly weaker binding to other DPA isomers. Importantly, mutations of conserved amino acid residues in the putative DPA(2,6)-binding pocket in SpoVAD essentially abolish its DPA(2,6)-binding capacity. Moreover, replacement of the wild-type spoVAD gene in B. subtilis with any of these spoVAD gene variants effectively eliminated DPA(2,6) uptake into developing spores in sporulation, although the variant proteins were still located in the spore inner membrane. Our results provide direct evidence that SpoVA proteins, in particular SpoVAD, are directly involved in DPA(2,6) movement into developing B. subtilis spores.