Annabel Parret

Clinical and Mutational Spectrum of Neurofibromatosis Type 1–like Syndrome

CONTEXT Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation. OBJECTIVE To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients. DESIGN, SETTING, AND PARTICIPANTS In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis. MAIN OUTCOME MEASURES Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations. RESULTS Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS. CONCLUSIONS A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1.

Proteasomal and Genetic Inactivation of the NF1 Tumor Suppressor in Gliomagenesis

Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However, complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity.

Bacteria killing their own kind: novel bacteriocins of Pseudomonas and other γ-proteobacteria

Abstract Pseudomonas aeruginosa strains frequently produce proteinaceous, narrow-spectrum antibacterial bacteriocins known as pyocins. The majority of these compounds exert their toxicity through non-specific DNA degradation inside sensitive cells. Here, we describe the identification of novel bacteriocins of different types in related bacteria inhabiting diverse niches. The abundance of bacteriocin genes in bacteria such as Pseudomonas , Klebsiella and Photorhabdus suggests that these systems play an important role in the competition between rival bacteria. The chimeric nature of these proteins implies that extensive domain swapping has contributed to the diversification of bacteriocins in γ-proteobacteria.

A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes

The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications.

Plant Lectin-Like Bacteriocin from a Rhizosphere-Colonizing Pseudomonas Isolate

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.

Novel lectin-like bacteriocins of biocontrol strain Pseudomonas fluorescens Pf-5.

ABSTRACT Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185:897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA-like genes, named llpA1Pf-5 and llpA2Pf-5. Recombinant Escherichia coli cells expressing llpA1Pf-5 or llpA2Pf-5 acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpABW11M1 antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1Pf-5 and LlpA2Pf-5 are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii. LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.

The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction

The brain‐specific synaptic guanosine triphosphatase (GTPase)‐activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide‐binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2–GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 × 104. The C2–GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.

Specific chaperones for the type VII protein secretion pathway

Background: Pathogenic mycobacteria use the type VII secretion systems (T7SS) ESX-1 and ESX-5 to secrete virulence factors, but it is unknown how these systems recognize their cognate substrates. Results: Pulldowns identified specific interactions between cytosolic components of ESX-1 and ESX-5 and subsets of cognate substrates. Conclusion: T7SS substrates interact with associated cytosolic secretion system components. Significance: Cytosolic chaperones contribute to system specificity in T7SS. Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG5, which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG5 interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG1 interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG5 and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG5 and EspG1 interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.

Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25-PPE41 dimer.

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX‐1, ESX‐3 and ESX‐5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system‐specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX‐5‐secreted PE25–PPE41 heterodimer in complex with the cytoplasmic chaperone EspG5. EspG5 represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG5‐binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone‐binding sequence, the hh motif, which is highly conserved among ESX‐1‐, ESX‐3‐ and ESX‐5‐specific PPE proteins. Disrupting the interaction between EspG5 and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG5 chaperone plays an important role in the ESX secretion mechanism by keeping aggregation‐prone PE–PPE proteins in their soluble state.

Novel bacteriocins with predicted tRNase and pore‐forming activities in Pseudomonas aeruginosa PAO1

Sir, Bacteriocins produced by Escherichia coli have been grouped into two major families, namely the nuclease and pore-forming colicins. In the nuclease colicins, the colicin E2 and colicin E3 subfamilies can be distinguished based on their DNase and RNase activities respectively. Pore-forming colicins are divided into the colicin E1 subfamily, the colicin A subfamily and the colicin Ia/Ib subfamily (Braun et al., 1994, Arch Microbiol 161: 199±206). The nuclease domain of the RNase colicin E5 exhibits no sequence similarity to those of the E3 subfamily colicins. Colicin E5 has recently been shown to hydrolyse transfer RNAs for Tyr, His, Asn and Asp on the 38 side of the nucleotide containing the modi®ed base queuine, found at the wobble position of each anticodon (Ogawa et al., 1999, Science 283: 2097±2100). A colicinogenic strain acquires speci®c immunity towards its bacteriocin through the production of an immunity protein that usually interacts with the C-terminal domain of the bacteriocin (Kleanthous et al., 1998, Mol Microbiol 28: 227±233). Both killing and immunity proteins are encoded by closely linked plasmidencoded genes. This genetic organization is also found in the chromosomally encoded S-type pyocins produced by Pseudomonas aeruginosa. Hitherto, the genes for four S-type pyocins (S1, S2, S3 and AP41) have been characterized in different P. aeruginosa strains (Sano et al., 1993, J Bacteriol 175: 2907±2916; Sano and Kageyama, 1993, Mol Gen Genet 237: 161±170; Duport et al., 1995, J Biol Chem 270: 8920±8927). Each of these pyocins is produced as a protein complex consisting of a killing protein with DNase activity and an immunity protein (Fig. 1). The killing proteins are organized in functional domains similar to the E2 colicins. In these colicins, the N-terminal region, involved in translocation, is followed by a receptor recognition domain and a DNase/immunity protein-binding domain. In pyocins, the order of receptor recognition and translocation domains is reversed (Fig. 1). The nuclease domains in pyocins S1, S2 and AP41 share about 50% identity with the nuclease domains of the E2 colicins (Kageyama et al., 1996, J Bacteriol 178: 103±110). The C-terminal domain of pyocin S3 and its immunity protein are unrelated to the nuclease domains and cognate immunity proteins of the other pyocins. Pyocins S2, S3 and AP41 contain an additional domain of unknown function (but dispensable for killing) linking the receptor-binding and translocation domains (Sano et al., 1993, J Bacteriol 175: 6179±6185). The availability of the P. aeruginosa PAO1 genomic sequence prompted us to search for additional pyocin genes. Homology searches with the four S-type pyocins and the cognate immunity proteins con®rmed the presence of the S2 pyocin gene, but also revealed a novel pyocin gene. The deduced protein sequence (764 amino acids) shows the highest level of overall homology with pyocin S3 (71% identity). Similar to pyocins AP41, S2 and S3, this new pyocin (designated pyocin S4) is composed of four domains. However, pyocin S4 has a novel composite domain structure (Fig. 1). Surprisingly, its C-terminal domain does not show homology with the corresponding regions of the known S pyocins, but with the C-terminal region of the tRNase colicin E5 (Fig. 1). The conserved operon structure of other pyocin genes was also found for pyocin S4 (Fig. 1). Consistent with the identi®cation of a colicin E5-like tRNase domain in pyocin S4, its putative immunity protein (112 amino acids) showed signi®cant homology with the immunity protein of colicin E5. The P. aeruginosa PAO1 genome appears to encode yet another novel type of pyocin and linked immunity protein, pyocin S5 (498 amino acids), containing only a short region with high homology to pyocin S2 (Fig. 1). The Cterminal domain is homologous to the active domain of the pore-forming colicins Ia and Ib. These colicins kill bacterial cells by the formation of voltage-gated, ionconducting channels across the plasma membrane (Wiener et al., 1997, Nature 385: 461±464). Downstream of the pyocin S5 gene, we identi®ed an open reading frame (ORF) for the probable immunity protein (108 amino acids), showing signi®cant homology with the colicins Ia and Ib immunity proteins (Fig. 1). The presence of three potential transmembrane helices in the pyocin S5 immunity protein (TMpred; http://www.ch.embnet.org/ software/TMPRED_ form.html) is consistent with the predicted channel-forming activity of the S5 pyocin. Immunity proteins for pore-forming colicins reside in the inner membrane, enabling speci®c intramembrane helix±helix interactions with the toxins (Cramer et al., 1995, Annu Rev Biophys Biomol Struct 24: 611±641; Espesset et al., 1996, EMBO J 15: 2356±2364). A P box, a conserved regulatory sequence found in the Molecular Microbiology (2000) 35(2), 472±475