Secretory expression of recombinant small laccase genes in Gram-positive bacteria.

BACKGROUND: Laccases are multicopper enzymes that oxidize a wide range of aromatic and non-aromatic compounds in the presence of oxygen. The majority of industrially relevant laccases are derived from fungi and are produced in eukaryotic expression systems such as Pichia pastoris and Saccharomyces cerevisiae. Bacterial laccases for research purposes are mostly produced intracellularly in Escherichia coli, but secretory expression systems are needed for future applications. Bacterial laccases from Streptomyces spp. are of interest for potential industrial applications because of their lignin degrading activities. RESULTS: In this study, we expressed small laccases genes from Streptomyces coelicolor, Streptomyces viridosporus and Amycolatopsis 75iv2 with their native signal sequences in Gram-positive Bacillus subtilis and Streptomyces lividans host organisms. The extracellular activities of ScLac, SvLac and AmLac expressed in S. lividans reached 1950 ± 99 U/l, 812 ± 57 U/l and 12 ± 1 U/l in the presence of copper supplementation. The secretion of the small laccases was irrespective of the copper supplementation; however, activities upon reconstitution with copper after expression were significantly lower, indicating the importance of copper during laccase production. The production of small laccases in B. subtilis resulted in extracellular activity that was significantly lower than in S. lividans. Unexpectedly, AmLac and ScLac were secreted without their native signal sequences in B. subtilis, indicating that B. subtilis secretes some heterologous proteins via an unknown pathway. CONCLUSIONS: Small laccases from S. coelicolor, S. viridosporus and Amycolatopsis 75iv2 were secreted in both Gram-positive expression hosts B. subtilis and S. lividans, but the extracellular activities were significantly higher in the latter.

Molecular cloning and functional analysis of HnSaratin from Hirudo nipponia.

In order to finish a bloodmeal successfully, hematophagous organisms often stored a variety of anticoagulant proteins in their salivary glands, such as proteins that inhibit platelet aggregation. When they ingest a bloodmeal, these proteins are injected into the host to prevent the blood from clotting. As one of the origins of leeches used in traditional Chinese medicine, H. nipponia was proved to be clinically effective in treatment of cardiovascular and cerebrovascular diseases. This study cloned the sequence of HnSaratin cDNA derived from salivary glands of H. nipponia. The sequence contains an open reading frame of 387 bp, encoding a protein of 128 amino acids containing a signal peptide of 21 amino acids. After removal of the signal peptide, the molecular mass of mature HnSaratin was 12.37 kDa, with a theoretical isoelectric point (pI) of 3.89. The N-terminal of mature HnSaratin was folded into a globular structure, in which 3 disulfide bonds, a ßßαßßß topology and 2 Glu residues that binds collagenous Lys2 were located, and the C-terminal formed a flexible region. The fusion HnSaratin protein was obtained by a prokaryotic expression system. The protein showed anti-platelet aggregation activity, and was observed to prevent blood clotting in rats. The significant high expression of HnSaratin mRNA in salivary glands was induced by bloodmeal ingestion of H. nipponia. Briefly, our work provides theoretical basis for further development and utilization of H. nipponia.

Characterization and Expression Analysis of Regeneration-Associated Protein (Aj-Orpin) during Intestinal Regeneration in the Sea Cucumber Apostichopus japonicus.

Apostichopus japonicus achieves intestinal regeneration in a short period after evisceration, and multiple genes are involved in this process. The transcriptome of A. japonicus was screened for regeneration-associated protein (Aj-Orpin), a gene that is specifically upregulated during intestinal regeneration. The expression and function of Aj-Orpin were identified and investigated in this study. The 5' and 3' RACE polymerase chain reaction (PCR) was used to clone the full-length cDNA of Aj-Orpin. The open reading frame codes for a 164 amino-acid protein with an EF-hand_7 domain and overlapping signal peptides and transmembrane regions. Moreover, Aj-Orpin mRNA and protein expression during intestinal regeneration was investigated using real-time quantitative PCR and Western blot. The expression pattern of Aj-Orpin in the regenerating intestine was investigated using immunohistochemistry. The results showed that Aj-Orpin is an exocrine protein with two EF-hand-like calcium-binding domains. Expression levels were higher in the regenerating intestine than in the normal intestine, but protein expression changes lagged behind mRNA expression changes. Aj-Orpin was found to play a role in the formation of blastema and lumen. It was primarily expressed in the serosal layer and submucosa, suggesting that it might be involved in proliferation. These observations lay the foundation for understanding the role of Orpin-like in echinoderm intestinal regeneration.

Genome-wide identification, characterization of Serotonin N-acetyltransferase and deciphering its importance under development, biotic and abiotic stress in soybean.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme involved in plant melatonin biosynthesis. Identifying its expression under development and stress will reveal the regulatory role in the soybean. To identify and characterize SNAT, we employed genome-wide analysis, gene structure, cis-acting elements, expression, and enzyme activity. We identified seven putative genes by genome-wide analysis and found chloroplast signal peptides in three GmSNATs. To elucidate GmSNATs role, expression datasets of more than a hundred samples related to circadian rhythm, developmental stages, and stress conditions were analysed. Notably, the expression of GmSNAT1 did not show significant expression during biotic and abiotic stress. The GmSNAT1 sequence showed 67.8 and 72.2 % similarities with OsSNAT and AtSNAT, respectively. The Km and Vmax of the purified recombinant GmSNAT1 were 657 µM and 3780 pmol/min/mg, respectively. To further understand the GmSNAT1 role, we supplemented different concentrations of serotonin and melatonin to in-vitro cultures and seed priming. These studies revealed that the GmSNAT1 expression was significantly up-regulated at higher concentrations of serotonin and down-regulated at higher melatonin concentrations. We speculate that a high concentration of melatonin during abiotic, biotic stress, and in-vitro cultures are responsible for regulating GmSNAT1 expression, which may regulate them at the enzyme level during stress in soybean.

cDNA Characterization and Expression of Selenium-Dependent CqGPx3 Isoforms in the Crayfish Cherax quadricarinatus under High Temperature and Hypoxia.

Glutathione peroxidase 3 (GPx3) is the only extracellular selenoprotein (Sel) that enzymatically reduces H2O2 to H2O and O2. Two GPx3 (CqGPx3) cDNAs were characterized from crayfish Cherax quadricarinatus. The nerve cord CqGPx3a isoform encodes for a preprotein containing an N-terminal signal peptide of 32 amino acid residues, with the mature Sel region of 192 residues and a dispensable phosphorylation domain of 36 residues. In contrast, the pereiopods CqGPx3b codes for a precursor protein with 19 residues in the N-terminal signal peptide, then the mature 184 amino acid residues protein and finally a Pro-rich peptide of 42 residues. CqGPx3 are expressed in cerebral ganglia, pereiopods and nerve cord. CqGPx3a is expressed mainly in cerebral ganglia, antennulae and nerve cord, while CqGPx3b was detected mainly in pereiopods. CqGPx3a expression increases with high temperature and hypoxia; meanwhile, CqGPx3b is not affected. We report the presence and differential expression of GPx3 isoforms in crustacean tissues in normal conditions and under stress for high temperature and hypoxia. The two isoforms are tissue specific and condition specific, which could indicate an important role of CqGPx3a in the central nervous system and CqGPx3b in exposed tissues, both involved in different responses to environmental stressors.

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications.

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.

Essentially Leading Antibody Production: An Investigation of Amino Acids, Myeloma, and Natural V-Region Signal Peptides in Producing Pertuzumab and Trastuzumab Variants.

Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody V-genes and their roles in recombinant transient production, systematically comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants. We found that amino acids count and types (essential or non-essential) were important factors in a logistic regression equation model for predicting transient co-transfection protein production rates. Deeper analysis revealed that the culture media were often incomplete and that the supplementation of essential amino acids can improve the recombinant protein yield. While these findings are derived from transient HEK293 expression, they also provide insights to the usage of the large repertoire of antibody signal peptides, where by varying the number of specific amino acids in the signal peptides attached to the variable regions, bottlenecks in amino acid availability can be mitigated.

Prediction of Peroxisomal Matrix Proteins in Plants.

Our knowledge of the proteome of plant peroxisomes is far from being complete, and the functional complexity and plasticity of this cell organelle are amazingly high particularly in plants, as exemplified by the model species Arabidopsis thaliana. Plant-specific peroxisome functions that have been uncovered only recently include, for instance, the participation of peroxisomes in phylloquinone and biotin biosynthesis. Experimental proteome studies have been proved very successful in defining the proteome of Arabidopsis peroxisomes but this approach also faces significant challenges and limitations. Complementary to experimental approaches, computational methods have emerged as important powerful tools to define the proteome of soluble matrix proteins of plant peroxisomes. Compared to other cell organelles such as mitochondria, plastids and the ER, the simultaneous operation of two major import pathways for soluble proteins in peroxisomes is rather atypical. Novel machine learning prediction approaches have been developed for peroxisome targeting signals type 1 (PTS1) and revealed high sensitivity and specificity, as validated by in vivo subcellular targeting analyses in diverse transient plant expression systems. Accordingly, the algorithms allow the correct prediction of many novel peroxisome-targeted proteins from plant genome sequences and the discovery of additional organelle functions. In contrast, the prediction of PTS2 proteins largely remains restricted to genome searches by conserved patterns contrary to more advanced machine learning methods. Here, we summarize and discuss the capabilities and accuracies of available prediction algorithms for PTS1 and PTS2 carrying proteins.

Synthetic Phosphorus Metabolic Pathway for Biosafety and Contamination Management of Cyanobacterial Cultivation.

Recent progress in genetic engineering and synthetic biology have greatly expanded the production capabilities of cyanobacteria, but concerns regarding biosafety issues and the risk of contamination of cultures in outdoor culture conditions remain to be resolved. With this dual goal in mind, we applied the recently established biological containment strategy based on phosphite (H3PO3, Pt) dependency to the model cyanobacterium Synechococcus elongatus PCC 7942 ( Syn 7942). Pt assimilation capability was conferred on Syn 7942 by the introduction of Pt dehydrogenase (PtxD) and hypophosphite transporter (HtxBCDE) genes that allow the uptake of Pt, but not phosphate (H3PO4, Pi). We then identified and disrupted the two indigenous Pi transporters, pst (Synpcc7942_2441 to 2445) and pit (Synpcc7942_0184). The resultant strain failed to grow on any media containing various types of P compounds other than Pt. The strain did not yield any escape mutants for at least 28 days with a detection limit of 3.6 × 10-11 per colony forming unit, and rapidly lost viability in the absence of Pt. Moreover, growth competition of the Pt-dependent strain with wild-type cyanobacteria revealed that the Pt-dependent strain could dominate in cultures containing Pt as the sole P source. Because Pt is rarely available in aquatic environments this strategy can contribute to both biosafety and contamination management of genetically engineered cyanobacteria.

Secretory overproduction of a raw starch-degrading glucoamylase in Penicillium oxalicum using strong promoter and signal peptide.

Raw starch-degrading enzymes (RSDEs) are capable of directly degrading raw starch granules below the gelatinization temperature of starch, which may significantly reduce the cost of starch-based biorefining. However, low yields of natural RSDEs from filamentous fungi limit their industrial application. In this study, transcriptomic and secretomic profiling was employed to screen strongest promoters and signal peptides for use in overexpression of a RSDE gene in Penicillium oxalicum. Top five strong promoters and three signal peptides were detected. Using a green fluorescent protein (GFP) as the reporter, the inducible promoter pPoxEgCel5B of an endoglucanase gene PoxEgCel5B and the signal peptide spPoxGA15A of a raw starch-degrading glucoamylase PoxGA15A were respectively identified as driving the highest GFP production in P. oxalicum. PoxGA15A-overexpressed P. oxalicum strain OXPoxGA15A, which was constructed based on both pPoxEgCel5B and spPoxGA15A, produced significantly higher amounts of recombinant PoxGA15A than the parental strain ∆PoxKu70. Furthermore, crude enzyme from the OXPoxGA15A strain exhibited high activities towards raw starch from cassava, potato, and uncooked soluble starch. Specifically, raw cassava starch-degrading enzyme activity reached 241.6 U/mL in the OXPoxGA15A, which was 3.4-fold higher than that of the ∆PoxKu70. This work provides a feasible method for hyperproduction of RSDEs in P. oxalicum.

TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells.

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of the mitochondrial enzyme, methylmalonyl-CoA mutase (MCM). The main treatments for MMA patients are dietary restriction of propiogenic amino acids and carnitine supplementation. Liver or combined liver/kidney transplantation has been used to treat those with the most severe clinical manifestations. Thus, therapies are necessary to help improve quality of life and prevent liver, renal and neurological complications. Previously, we successfully used the TAT-MTS-Protein approach for replacing a number of mitochondrial-mutated proteins. In this targeted system, TAT, an 11 a.a peptide, which rapidly and efficiently can cross biological membranes, is fused to a mitochondrial targeting sequence (MTS), followed by the mitochondrial mature protein which sends the protein into the mitochondria. In the mitochondria, the TAT-MTS is cleaved off and the native protein integrates into its natural complexes and is fully functional. In this study, we used heterologous MTSs of human, nuclear-encoded mitochondrial proteins, to target the human MCM protein into the mitochondria. All fusion proteins reached the mitochondria and successfully underwent processing. Treatment of MMA patient fibroblasts with these fusion proteins restored mitochondrial activity such as ATP production, mitochondrial membrane potential and oxygen consumption, indicating the importance of mitochondrial function in this disease. Treatment with the fusion proteins enhanced cell viability and most importantly reduced MMA levels. Treatment also enhanced albumin and urea secretion in a CRISPR/Cas9-engineered HepG2 MUT (-/-) liver cell line. Therefore, we suggest using this TAT-MTS-Protein approach for the treatment of MMA.

PPero, a Computational Model for Plant PTS1 Type Peroxisomal Protein Prediction.

Well-defined motifs often make it easy to investigate protein function and localization. In plants, peroxisomal proteins are guided to peroxisomes mainly by a conserved type 1 (PTS1) or type 2 (PTS2) targeting signal, and the PTS1 motif is commonly used for peroxisome targeting protein prediction. Currently computational prediction of peroxisome targeted PTS1-type proteins are mostly based on the 3 amino acids PTS1 motif and the adjacent sequence which is less than 14 amino acid residue in length. The potential contribution of the adjacent sequences beyond this short region has never been well investigated in plants. In this work, we develop a bi-profile Bayesian SVM method to extract and learn position-based amino acid features for both PTS1 motifs and their extended adjacent sequences in plants. Our proposed model outperformed other implementations with similar applications and achieved the highest accuracy of 93.6% and 92.6% for Arabidosis and other plant species respectively. A large scale analysis for Arabidopsis, Rice, Maize, Potato, Wheat, and Soybean proteome was conducted using the proposed model and a batch of candidate PTS1 proteins were predicted. The DNA segments corresponding to the C-terminal sequences of 9 selected candidates were cloned and transformed into Arabidopsis for experimental validation, and 5 of them demonstrated peroxisome targeting.

Transesterification of plant oils using Staphylococcus haemolyticus L62 lipase displayed on Escherichia coli cell surface using the OmpA signal peptide and EstAß8 anchoring motif.

Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the outer membrane of Escherichia coli using the OmpA signal peptide and the autotransporter EstAß8 protein. Localization of SHL62 lipase on the outer membrane of E. coli was confirmed using immunofluorescence microscopy and flow cytometry analysis. Lipase activity of the displayed SHL62 lipase was also measured using spectrophotometry and pH titration. SHL62 lipase activity of whole cells reached 2.0U/ml culture (OD600nm of 10) when it was measured by the p-nitrophenyl caprylate assay after being induced with 1mM IPTG for 24h. The optimum temperature and pH for the lipase was 45°C and 10, respectively. Furthermore, it maintained more than 90% of maximum lipase activity at up to 50°C and in a pH range of 5-9. The hydrolytic activity assay conduted with various substrates confirmed that p-nitrophenyl caprylate and corn oil were preferred substrates among various synthetic and natural substrates, respectively. The displayed SHL62 lipase produced fatty acid esters from various alcohols and plant oils through transesterification.

Ectopic expression of human acidic fibroblast growth factor 1 in the medicinal plant, Salvia miltiorrhiza, accelerates the healing of burn wounds.

BACKGROUND: Healing of burns is a complex process and very few effective treatments exist to facilitate the burn recovery process. Human acidic fibroblast growth factor 1 (FGF-1) plays an important role in a variety of biological processes, including angiogenesis, and tissue repair. Salvia miltiorrhiza is widely used in traditional Chinese medicine as an herb for the treatment of various diseases, including cardiovascular and cerebrovascular diseases, and traumatic injuries. We present that expression of FGF-1 in S. miltiorrhiza significantly accelerates the healing of burn wounds. RESULTS: The human fgf-1 gene was fused with a barley α-amylase signal peptide DNA sequence and driven by a 35S promoter for constitutive expression in transgenic S. miltiorrhiza plants. The highest yield of recombinant FGF-1 obtained from leaves of transgenic S. miltiorrhiza lines was 272 ng/fresh weight. Aqueous extracts from transgenic S. miltiorrhiza exhibited FGF-1 activity approximately 19.2-fold greater than that of the standard FGF-1. Compared to the standard FGF-1 or the extracts obtained from non-transgenic plants, it stimulated proliferation of Balb/c 3 T3 mouse fibroblast cells assessed with the standard MTT assay and promoted angiogenesis in the chicken embryo chorioallantoic membrane (CAM) assay. Topical application of the extract significantly accelerated the burn wound healing process. CONCLUSIONS: The product appears to retain the biological activity of both FGF-1 as well as the medicinal properties of the plant. The extracts from transgenic S. miltiorrhiza combines the therapeutic functions of FGF-1 and the medicinal plant, S. miltiorrhiza. Topical application of the product can reduce the costs associated with extraction, purification, and recovery.

Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology.

Towards the targeting of recombinant Thermoanaerobacter thermohydrosulfuricus lipase (TtL) for secretion into the culture medium of Escherichia coli, we have investigated a combination of the archeal lipase gene with a Salinovibrio metalloprotease (SVP2) signal peptide sequence. The SVP2 signal peptide has shown all necessary features of a leader sequence for high level secretion of a recombinant target protein in E. coli. Two sets of primers were designed for amplification of the corresponding gene fragments by PCR. Firstly, the PCR product of the TtL gene with designed restriction sites of SacI and HindIII was cloned into pQE-80L plasmid, named as pQE80L-TtL. Afterwards, the amplified fragment of SVP2 signal peptide with EcoRI and SacI restriction sites was also cloned into pQE80L-TtL and the final construct pQE-STL was obtained. A study on the extracellular expression of recombinant STL revealed that most of the enzyme activity was located in the periplasmic space. Glycine and Triton X-100 were investigated to determine whether the leakage of recombinant STL from the outer membrane was promoted, and it was revealed that glycine has a positive effect. Statistical media optimization design was then applied to optimize the effect of seven factors including glycine, Triton X-100, IPTG, yeast extract concentration, incubation time, induction time, and temperature on the extracellular expression of STL. The optimum conditions for the secretion of the lipase was obtained by incubating recombinant E. coli BL21 cells in the medium supplemented by 1.27% glycine and 24h of incubation in the presence of 0.2mM IPTG concentration.

Molecular characterization of a cDNA encoding red pigment-concentrating hormone in black tiger shrimp Penaeus monodon: Implication of its function in molt and osmoregulation.

Red pigment-concentrating hormone (RPCH) is a member of the AKH/RPCH peptide family present mainly in crustaceans and insects. Insect AKH is responsible for metabolic functions whereas RPCH plays a major role in the aggregation of red chromatophores in crustaceans. In this study, a full-length cDNA of RPCH of the black tiger shrimp, Penaeus monodon (PmRPCH) was cloned by Rapid Amplification of cDNA Ends strategies from the eyestalk RNA. A 770 bp full-length PmRPCH cDNA harbored 279 bp of an open reading frame encoding a signal peptide of 21 amino acid residues, an 8 amino acid mature RPCH peptide, followed by 61 amino acid residues of a RPCH precursor-related peptide. The highest levels of PmRPCH mRNA expression were detected in eyestalks while lower expression was found in other nervous tissues i.e. brain, thoracic ganglia and abdominal nerve cord. Expression of PmRPCH was transiently stimulated upon hypersalinity change within 12 h suggesting its osmoregulatory function. During the molting cycle, PmRPCH in the eyestalk was expressed at the lowest level in the early pre-molt stage (D0), then gradually increased over the pre-molt period and reached the highest level in the late pre-molt (D4) and post-molt (AB) stages. RPCH peptide at a dose of 100 pmol also increased gill Na(+)/K(+) ATPase activity in 36-48 h after injection. However, PmRPCH did not accelerate the duration of molting cycle. Our results provide the first evidence on the potential function of PmRPCH in molting, probably by mediating hemolymph osmolality and ion transport enzymes during the late pre-molt stage.

Identification of new protein coding sequences and signal peptidase cleavage sites of Helicobacter pylori strain 26695 by proteogenomics.

Correct annotation of protein coding genes is the basis of conventional data analysis in proteomic studies. Nevertheless, most protein sequence databases almost exclusively rely on gene finding software and inevitably also miss protein annotations or possess errors. Proteogenomics tries to overcome these issues by matching MS data directly against a genome sequence database. Here we report an in-depth proteogenomics study of Helicobacter pylori strain 26695. MS data was searched against a combined database of the NCBI annotations and a six-frame translation of the genome. Database searches with Mascot and X! Tandem revealed 1115 proteins identified by at least two peptides with a peptide false discovery rate below 1%. This represents 71% of the predicted proteome. So far this is the most extensive proteome study of Helicobacter pylori. Our proteogenomic approach unambiguously identified four previously missed annotations and furthermore allowed us to correct sequences of six annotated proteins. Since secreted proteins are often involved in pathogenic processes we further investigated signal peptidase cleavage sites. By applying a database search that accommodates the identification of semi-specific cleaved peptides, 63 previously unknown signal peptides were detected. The motif LXA showed to be the predominant recognition sequence for signal peptidases. BIOLOGICAL SIGNIFICANCE: The results of MS-based proteomic studies highly rely on correct annotation of protein coding genes which is the basis of conventional data analysis. However, the annotation of protein coding sequences in genomic data is usually based on gene finding software. These tools are limited in their prediction accuracy such as the problematic determination of exact gene boundaries. Thus, protein databases own partly erroneous or incomplete sequences. Additionally, some protein sequences might also be missing in the databases. Proteogenomics, a combination of proteomic and genomic data analyses, is well suited to detect previously not annotated proteins and to correct erroneous sequences. For this purpose, the existing database of the investigated species is typically supplemented with a six-frame translation of the genome. Here, we studied the proteome of the major human pathogen Helicobacter pylori that is responsible for many gastric diseases such as duodenal ulcers and gastric cancer. Our in-depth proteomic study highly reliably identified 1115 proteins (FDR<0.01%) by at least two peptides (FDR<1%) which represent 71% of the predicted proteome deposited at NCBI. The proteogenomic data analysis of our data set resulted in the unambiguous identification of four previously missed annotations, the correction of six annotated proteins as well as the detection of 63 previously unknown signal peptides. We have annotated proteins of particular biological interest like the ferrous iron transport protein A, the coiled-coil-rich protein HP0058 and the lipopolysaccharide biosynthesis protein HP0619. For instance, the protein HP0619 could be a drug target for the inhibition of the LPS synthesis pathway. Furthermore it has been proven that the motif "LXA" is the predominant recognition sequence for the signal peptidase I of H. pylori. Signal peptidases are essential enzymes for the viability of bacterial cells and are involved in pathogenesis. Therefore signal peptidases could be novel targets for antibiotics. The inclusion of the corrected and new annotated proteins as well as the information of signal peptide cleavage sites will help in the study of biological pathways involved in pathogenesis or drug response of H. pylori.

Byssal proteins of the freshwater zebra mussel, Dreissena polymorpha.

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.

Avirulence protein 3a (AVR3a) from the potato pathogen Phytophthora infestans forms homodimers through its predicted translocation region and does not specifically bind phospholipids.

The mechanism of translocation of RxLR effectors from plant pathogenic oomycetes into the cytoplasm of their host is currently the object of intense research activity and debate. Here, we report the biochemical and thermodynamic characterization of the Phytophthora infestans effector AVR3a in vitro. We show that the amino acids surrounding the RxLR leader mediate homodimerization of the protein. Dimerization was considerably attenuated by a localized mutation within the RxLR motif that was previously described to prevent translocation of the protein into host. Importantly, we confirm that the reported phospholipid-binding properties of AVR3a are mediated by its C-terminal effector domain, not its RxLR leader. However, we show that the observed phospholipid interaction is attributable to a weak association with denatured protein molecules and is therefore most likely physiologically irrelevant.

Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7.

The import of a subset of peroxisomal matrix proteins is mediated by the peroxisomal targeting signal 2 (PTS2). The results of our sequence and physical property analysis of known PTS2 signals and of a mutational study of the least characterized amino acids of a canonical PTS2 motif indicate that PTS2 forms an amphipathic helix accumulating all conserved residues on one side. Three-dimensional structural modeling of the PTS2 receptor PEX7 reveals a groove with an evolutionarily conserved charge distribution complementary to PTS2 signals. Mammalian two-hybrid assays and cross-complementation of a mutation in PTS2 by a compensatory mutation in PEX7 confirm the interaction site. An unstructured linker region separates the PTS2 signal from the core protein. This additional information on PTS2 signals was used to generate a PTS2 prediction algorithm that enabled us to identify novel PTS2 signals within human proteins and to describe KChIP4 as a novel peroxisomal protein.