Evaluating the Impact of Different Sequence Databases on Metaproteome Analysis: Insights from a Lab-Assembled Microbial Mixture

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge.This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.     

Lesion of caudate-putamen interneurons with kainic acid alters dopamine and serotonin metabolism in the olfactory tubercle of the rat

1. The existence of functional interrelationships between dorsal and ventral regions of the rat striatum was investigated. Kainic acid (KA) was employed to induce neuronal lesions in the more dorsal striatum, the caudate-putamen (CP). Only one CP (one side) received KA. KA-induced neurotoxicity at the site of injection (CP) was evidenced by reductions in choline-acetyltransferase activity and in GABA levels, and by increases in the ratios metabolite/monoamine for dopamine (DA) and serotonin (5-HT).2. In addition to the well-known local effects, direct stereotaxic injection of KA into the CP produced distant effects in the ipsilateral olfactory tubercle (OT). A dose-dependent increase in the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) and decreases in DA and 5-HT concentrations were observed in the OT ipsilateral to the CP injected with KA. With 1, 2, 3, and 4 g of KA, the ratio DOPAC+HVA/DA in the OT was 30, 79, 140, and 173% higher, respectively, than control levels. With 2, 3, and 4 g of KA, the levels of 5-HIAA were approximately 30, 60, and 120% higher than control values, and the changes in 5-HIAA were associated with significant reductions in 5-HT concentrations.3. Our results suggest that the dorsal part of the striatum exerts important regulatory functions over the most ventral striatal region, the OT. Destruction of CP interneurons by KA leads to disinhibition of DA and 5-HT activities to the OT. The functional interactions between dorsal and ventral striatal regions may play a role in the integration of fundamental life-preserving, motivational, and goal-directed olfactory motor behaviors of rodents.     

Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein

Information processing pathways such as DNA replication are conserved in eukaryotes and archaea and are significantly different from those found in bacteria. Single-stranded DNA-binding (SSB) proteins (or replication protein A, RPA, in eukaryotes) play a central role in many of these pathways. However, whilst euryarchaea have a eukaryotic-type RPA homologue, crenarchaeal SSB proteins appear much more similar to the bacterial proteins, with a single OB fold for DNA binding and a flexible C-terminal tail that is implicated in protein-protein interactions. We have determined the crystal structure of the SSB protein from the crenarchaeote Sulfolobus solfataricus to 1.26 A. The structure shows a striking and unexpected similarity to the DNA-binding domains of human RPA, providing confirmation of the close relationship between archaea and eukaryotes. The high resolution of the structure, together with thermodynamic and mutational studies of DNA binding, allow us to propose a molecular basis for DNA binding and define the features required for eukaryotic and archaeal OB folds.     

Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [(15)N]NH(4)Cl and [(13)C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

Contribution of DEAF1 structural domains to the interaction with the breast cancer oncogene LMO4

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1.     

Equine and Canine Anaplasma phagocytophilum Strains Isolated on the Island of Sardinia (Italy) Are Phylogenetically Related to Pathogenic Strains from the United States

The presence of Anaplasma phagocytophilum, a tick-transmitted zoonotic pathogen, was investigated in Sardinia using a molecular approach. Phylogenetic analysis revealed that Sardinian strains are genetically distinct from the two lineages previously described in Europe and are closely related to strains isolated in different areas of the United States.     

NMR assignment of prespore specific antigen—a cell surface adhesion glycoprotein from Dictyostelium discoideum

Presopore-specific antigen (PsA) is a cell surface glycoprotein of the cellular slime mould Dictyostelium discoidum implicated in cell adhesion. The ¹⁵N, ¹³C and ¹H chemical shift assignments of PsA were determined from multidimensional, multinuclear NMR experiments. Resonance assignments have been made for both the N-terminal globular domain and its attached O-glycosylated PTVT linker motif.     

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

Backbone <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of the OB domain of the single stranded DNA-binding protein hSSB1 (NABP2/OBFC2B) and chemical shift mapping of the DNA-binding interface

Single-stranded DNA-binding proteins (SSBs) are highly important in DNA metabolism and play an essential role in all major DNA repair pathways. SSBs are generally characterised by the presence of an oligonucleotide binding (OB) fold which is able to recognise single-stranded DNA (ssDNA) with high affinity. We discovered two news SSBs in humans (hSSB1 and hSSB2) that both contain a single OB domain followed by a divergent spacer region and a charged C-terminus. We have extensively characterised one of these, hSSB1 (NABP2/OBFC2B), in numerous important DNA processing events such as, in DNA double-stranded break repair and in the response to oxidative DNA damage. Although the structure of hSSB1 bound to ssDNA has recently been determined using X-ray crystallography, the detailed atomic level mechanism of the interaction of hSSB1 with ssDNA in solution has not been established. In this study we report the solution-state backbone chemical shift assignments of the OB domain of hSSB1. In addition, we have utilized NMR to map the DNA-binding interface of hSSB1, revealing major differences between recognition of ssDNA under physiological conditions and in the recently determined crystal structure. Our NMR data in combination with further biophysical and biochemical experiments will allow us to address these discrepancies and shed light onto the structural basis of DNA-binding by hSSB1 in solution.