Profiling of Parkin-binding partners using tandem affinity purification.

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

Characterization of the EGFR interactome reveals associated protein complex networks and intracellular receptor dynamics.

Growth factor receptor mediated signaling is meanwhile recognized as a complex signaling network, which is initiated by recruiting specific patterns of adaptor proteins to the intracellular domain of epidermal growth factor receptor (EGFR). Approaches to globally identify EGFR-binding proteins are required to elucidate this network. We affinity-purified EGFR with its interacting proteins by coprecipitation from lysates of A431 cells. A total of 183 proteins were repeatedly detected in high-resolution MS measurements. For 15 of these, direct interactions with EGFR were listed in the iRefIndex interaction database, including Grb2, shc-1, SOS1 and 2, STAT 1 and 3, AP2, UBS3B, and ERRFI. The newly developed Cytoscape plugin ModuleGraph allowed retrieving and visualizing 93 well-described protein complexes that contained at least one of the proteins found to interact with EGFR in our experiments. Abundances of 14 proteins were modulated more than twofold upon EGFR activation whereof clathrin-associated adaptor complex AP-2 showed 4.6-fold enrichment. These proteins were further annotated with different cellular compartments. Finally, interactions of AP-2 proteins and the newly discovered interaction of CIP2A could be verified. In conclusion, a powerful technique is presented that allowed identification and quantitative assessment of the EGFR interactome to provide further insight into EGFR signaling.

Association between variants of PRDM1 and NDP52 and Crohn's disease, based on exome sequencing and functional studies.

BACKGROUND & AIMS: Genome-wide association studies (GWAS) have identified 140 Crohn's disease (CD) susceptibility loci. For most loci, the variants that cause disease are not known and the genes affected by these variants have not been identified. We aimed to identify variants that cause CD through detailed sequencing, genetic association, expression, and functional studies. METHODS: We sequenced whole exomes of 42 unrelated subjects with CD and 5 healthy subjects (controls) and then filtered single nucleotide variants by incorporating association results from meta-analyses of CD GWAS and in silico mutation effect prediction algorithms. We then genotyped 9348 subjects with CD, 2868 subjects with ulcerative colitis, and 14,567 control subjects and associated variants analyzed in functional studies using materials from subjects and controls and in vitro model systems. RESULTS: We identified rare missense mutations in PR domain-containing 1 (PRDM1) and associated these with CD. These mutations increased proliferation of T cells and secretion of cytokines on activation and increased expression of the adhesion molecule L-selectin. A common CD risk allele, identified in GWAS, correlated with reduced expression of PRDM1 in ileal biopsy specimens and peripheral blood mononuclear cells (combined P = 1.6 × 10(-8)). We identified an association between CD and a common missense variant, Val248Ala, in nuclear domain 10 protein 52 (NDP52) (P = 4.83 × 10(-9)). We found that this variant impairs the regulatory functions of NDP52 to inhibit nuclear factor κB activation of genes that regulate inflammation and affect the stability of proteins in Toll-like receptor pathways. CONCLUSIONS: We have extended the results of GWAS and provide evidence that variants in PRDM1 and NDP52 determine susceptibility to CD. PRDM1 maps adjacent to a CD interval identified in GWAS and encodes a transcription factor expressed by T and B cells. NDP52 is an adaptor protein that functions in selective autophagy of intracellular bacteria and signaling molecules, supporting the role of autophagy in the pathogenesis of CD.

Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.

Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

Genome-wide association study identifies a psoriasis susceptibility locus at TRAF3IP2.

Psoriasis is a multifactorial skin disease characterized by epidermal hyperproliferation and chronic inflammation, the most common form of which is psoriasis vulgaris (PsV). We present a genome-wide association analysis of 2,339,118 SNPs in 472 PsV cases and 1,146 controls from Germany, with follow-up of the 147 most significant SNPs in 2,746 PsV cases and 4,140 controls from three independent replication panels. We identified an association at TRAF3IP2 on 6q21 and genotyped two SNPs at this locus in two additional replication panels (the combined discovery and replication panels consisted of 6,487 cases and 8,037 controls; combined P = 2.36 × 10⁻¹° for rs13210247 and combined P = 1.24 × 10⁻¹6 for rs33980500). About 15% of psoriasis cases develop psoriatic arthritis (PsA). A stratified analysis of our datasets including only PsA cases (1,922 cases compared to 8,037 controls, P = 4.57 × 10⁻¹² for rs33980500) suggested that TRAF3IP2 represents a shared susceptibility for PsV and PsA. TRAF3IP2 encodes a protein involved in IL-17 signaling and which interacts with members of the Rel/NF-κB transcription factor family.

Structure-function analysis of the NB-ARC domain of plant disease resistance proteins.

Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

Identification of PatL1, a human homolog to yeast P body component Pat1.

In yeast, the activators of mRNA decapping, Pat1, Lsm1 and Dhh1, accumulate in processing bodies (P bodies) together with other proteins of the 5'-3'-deadenylation-dependent mRNA decay pathway. The Pat1 protein is of particular interest because it functions in the opposing processes of mRNA translation and mRNA degradation, thus suggesting an important regulatory role. In contrast to other components of this mRNA decay pathway, the human homolog of the yeast Pat1 protein was unknown. Here we describe the identification of two human PAT1 genes and show that one of them, PATL1, codes for an ORF with similar features as the yeast PAT1. As expected for a protein with a fundamental role in translation control, PATL1 mRNA was ubiquitously expressed in all human tissues as were the mRNAs of LSM1 and RCK, the human homologs of yeast LSM1 and DHH1, respectively. Furthermore, fluorescence-tagged PatL1 protein accumulated in distinct foci that correspond to P bodies, as they co-localized with the P body components Lsm1, Rck/p54 and the decapping enzyme Dcp1. In addition, as for its yeast counterpart, PatL1 expression was required for P body formation. Taken together, these data emphasize the conservation of important P body components from yeast to human cells.

Systematic association mapping identifies NELL1 as a novel IBD disease gene.

Crohn disease (CD), a sub-entity of inflammatory bowel disease (IBD), is a complex polygenic disorder. Although recent studies have successfully identified CD-associated genetic variants, these susceptibility loci explain only a fraction of the heritability of the disease. Here, we report on a multi-stage genome-wide scan of 393 German CD cases and 399 controls. Among the 116,161 single-nucleotide polymorphisms tested, an association with the known CD susceptibility gene NOD2, the 5q31 haplotype, and the recently reported CD locus at 5p13.1 was confirmed. In addition, SNP rs1793004 in the gene encoding nel-like 1 precursor (NELL1, chromosome 11p15.1) showed a consistent disease-association in independent German population- and family-based samples (942 cases, 1082 controls, 375 trios). Subsequent fine mapping and replication in an independent sample of 454 French/Canadian CD trios supported the authenticity of the NELL1 association. Further confirmation in a large German ulcerative colitis (UC) sample indicated that NELL1 is a ubiquitous IBD susceptibility locus (combined p<10(-6); OR = 1.66, 95% CI: 1.30-2.11). The novel 5p13.1 locus was also replicated in the French/Canadian sample and in an independent UK CD patient panel (453 cases, 521 controls, combined p<10(-6) for SNP rs1992660). Several associations were replicated in at least one independent sample, point to an involvement of ITGB6 (upstream), GRM8 (downstream), OR5V1 (downstream), PPP3R2 (downstream), NM_152575 (upstream) and HNF4G (intron).

A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.

We performed a genome-wide association study of 19,779 nonsynonymous SNPs in 735 individuals with Crohn disease and 368 controls. A total of 7,159 of these SNPs were informative. We followed up on all 72 SNPs with P 0.4), these data suggest that the underlying biological process may be specific to Crohn disease.

Mutations in the MutSalpha interaction interface of MLH1 can abolish DNA mismatch repair.

MutLalpha, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSalpha and assembles and controls further repair enzymes. We tested if the interaction of MutLalpha with DNA-bound MutSalpha is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLalpha for MutSalpha is located on the edge of an extensive beta-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein-protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL-MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLalpha-MutSalpha interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis.

Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

Mutations in the NB-ARC domain of I-2 that impair ATP hydrolysis cause autoactivation.

Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).

Diversity and functional plasticity of eukaryotic selenoproteins: identification and characterization of the SelJ family.

Selenoproteins are a diverse group of proteins that contain selenocysteine (Sec), the 21st amino acid. In the genetic code, UGA serves as a termination signal and a Sec codon. This dual role has precluded the automatic annotation of selenoproteins. Recent advances in the computational identification of selenoprotein genes have provided a first glimpse of the size, functions, and phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the identification of a selenoprotein family named SelJ. In contrast to known selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant similarity to the jellyfish J1-crystallins and with them constitutes a distinct subfamily within the large family of ADP-ribosylation enzymes. Consistent with its potential role as a structural crystallin, SelJ has preferential and homogeneous expression in the eye lens in early stages of zebrafish development. A structural role for SelJ would be in contrast to the majority of known selenoenzymes. The unusually highly restricted phylogenetic distribution of SelJ, its specialization, and the comparative analysis of eukaryotic selenoproteomes reveal the diversity and functional plasticity of selenoproteins and point to a mosaic evolution of the use of Sec in proteins.

Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD), Crohn disease (CD), and ulcerative colitis (UC) are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11), CD patients (n = 10) and UC patients (n = 10). 33P-radiolabeled cDNA from purified poly(A)+ RNA extracted from biopsies (unpooled) was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome) and CDH11 (cadherin 11, type 2). By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

Genetics of Crohn disease, an archetypal inflammatory barrier disease.

Chronic inflammatory disorders such as Crohn disease, atopic eczema, asthma and psoriasis are triggered by hitherto unknown environmental factors that function on the background of some polygenic susceptibility. Recent technological advances have allowed us to unravel the genetic aetiology of these and other complex diseases. Using Crohn disease as an example, we show how the discovery of susceptibility genes furthers our understanding of the underlying disease mechanisms and how it will, ultimately, give rise to new therapeutic developments. The long-term goal of such endeavours is to develop targeted prophylactic strategies. These will probably target the molecular interaction on the mucosal surface between the products of the genome and the microbial metagenome of a patient.

A new CARD15 mutation in Blau syndrome.

The caspase recruitment domain gene CARD15/NOD2, encoding a cellular receptor involved in an NF-kappaB-mediated pathway of innate immunity, was first identified as a major susceptibility gene for Crohn's disease (CD), and more recently, as responsible for Blau syndrome (BS), a rare autosomal-dominant trait characterized by arthritis, uveitis, skin rash and granulomatous inflammation. While CARD15 variants associated with CD are located within or near the C-terminal leucine-rich repeat domain and cause decreased NF-kappaB activation, BS mutations affect the central nucleotide-binding NACHT domain and result in increased NF-kappaB activation. In an Italian family with BS, we detected a novel mutation E383K, whose pathogenicity is strongly supported by cosegregation with the disease in the family and absence in controls, and by the evolutionary conservation and structural role of the affected glutamate close to the Walker B motif of the nucleotide-binding site in the NACHT domain. Interestingly, substitutions at corresponding positions in another NACHT family member cause similar autoinflammatory phenotypes.

Sarcoidosis is associated with a truncating splice site mutation in BTNL2.

Sarcoidosis is a polygenic immune disorder with predominant manifestation in the lung. Genome-wide linkage analysis previously indicated that the extended major histocompatibility locus on chromosome 6p was linked to susceptibility to sarcoidosis. Here, we carried out a systematic three-stage SNP scan of 16.4 Mb on chromosome 6p21 in as many as 947 independent cases of familial and sporadic sarcoidosis and found that a 15-kb segment of the gene butyrophilin-like 2 (BTNL2) was associated with the disease. The primary disease-associated variant (rs2076530; P(TDT) = 3 x 10(-6), P(case-control) = 1.1 x 10(-8); replication P(TDT) = 0.0018, P(case-control) = 1.8 x 10(-6)) represents a risk factor that is independent of variation in HLA-DRB1. BTNL2 is a member of the immunoglobulin superfamily and has been implicated as a costimulatory molecule involved in T-cell activation on the basis of its homology to B7-1. The G --> A transition constituting rs2076530 leads to the use of a cryptic splice site located 4 bp upstream of the affected wild-type donor site. Transcripts of the risk-associated allele have a premature stop in the spliced mRNA. The resulting protein lacks the C-terminal IgC domain and transmembrane helix, thereby disrupting the membrane localization of the protein, as shown in experiments using green fluorescent protein and V5 fusion proteins.

The HIN domain of IFI-200 proteins consists of two OB folds.

The interferon-inducible p200 (IFI-200/HIN-200) family of proteins regulates cell growth and differentiation, and confers resistance to the development of tumors and virus infections. IFI-200 family members are thought to exert their biological effects by modulation of the transcriptional activities of numerous factors and interaction with other proteins through the C-terminal HIN domains. However, the HIN domain structure and function have remained obscure. Therefore, we performed a comprehensive bioinformatics analysis and assembled a structure-based multiple sequence alignment of IFI-200 proteins. The application of fold recognition methods revealed that the HIN domain consists of two consecutive OB domains. Our structural models of DNA-binding HIN domains afford the long-sought interpretations for many previous experimental observations. Our results also raise the possibility of as yet unexplored functional roles of IFI-200 proteins as transcriptional regulators and as interaction partners of proteins involved in immunomodulatory and apoptotic processes.

Novel Sm-like proteins with long C-terminal tails and associated methyltransferases.

Sm and Sm-like proteins of the Lsm (like Sm) domain family are generally involved in essential RNA-processing tasks. While recent research has focused on the function and structure of small family members, little is known about Lsm domain proteins carrying additional domains. Using an integrative bioinformatics approach, we discovered five novel groups of Lsm domain proteins (Lsm12-16) with long C-terminal tails and investigated their functions. All of them are evolutionarily conserved in eukaryotes with an N-terminal Lsm domain to bind nucleic acids followed by as yet uncharacterized C-terminal domains and sequence motifs. Based on known yeast interaction partners, Lsm12-16 may play important roles in RNA metabolism. Particularly, Lsm12 is possibly involved in mRNA degradation or tRNA splicing, and Lsm13-16 in the regulation of the mitotic G2/M phase. Lsm16 proteins have an additional C-terminal YjeF_N domain of as yet unknown function. The identification of an additional methyltransferase domain at the C-terminus of one of the Lsm12 proteins also led to the recognition of three new groups of methyltransferases, presumably dependent on S-adenosyl-l-methionine. Further computational analyses revealed that some methyltransferases contain putative RNA-binding helix-turn-helix domains and zinc fingers.

Structural and functional analysis of ataxin-2 and ataxin-3.

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are autosomal-dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding regions of the genes encoding ataxin-2 and ataxin-3, respectively. To provide a rationale for further functional experiments, we explored the protein architectures of ataxin-2 and ataxin-3. Using structure-based multiple sequence alignments of homologous proteins, we investigated domains, sequence motifs, and interaction partners. Our analyses focused on presumably functional amino acids and the construction of tertiary structure models of the RNA-binding Lsm domain of ataxin-2 and the deubiquitinating Josephin domain of ataxin-3. We also speculate about distant evolutionary relationships of ubiquitin-binding UIM, GAT, UBA and CUE domains and helical ANTH and UBX domain extensions.