The plasmids found in isolates of the acidothermophilic archaebacterium Thermoplasma acidophilum

Abstract Plasmids were detected in isolates of an acidothermophilic archaebacterium Thermoplasma acidophilum . One of the plasmids, pTA1, was characterized. The plasmid was a circular DNA of 15.2 kbp. A physical map was constructed using three restriction endonucleases. A copy number of this plasmid was estimated to be 7–13 per cell. The homologous sequence was not found in the chromosomal DNA of the host cell.     

Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M(r) of 250,000. It consists of four, apparently identical subunits each of M(r) 60,000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.     

Proteomics Analysis of Thermoplasma Quinone Droplets

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin‐N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D‐SDS‐PAGE/LC–MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods—column chromatography, immunoprecipitation, and LD ultracentrifugation—are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.     

Low-angle X-ray scattering analysis of the Thermoplasma acidophilum nucleoprotein subunit

A DNA-protein complex isolated from Thermoplasma acidophilum has been examined using low-angle X-ray scattering measurements. In agreement with the results of electron-microscopic studies a diamter of 5.5 nm is deduced. Finally, a simplified model of the DNA-protein particles is discussed postulating a kinked DNA.     

嗜酸热原体菌的超氧化物歧化酶的结构模建和进化踪迹分析

为考察来源于极端高温酸性环境中的超氧化物歧化酶,建立了一个由嗜酸热原体茵的sod基因编码的蛋白质的可靠三维分子结构,并对这个蛋白质进行进化踪迹分析识别出了11个踪迹残基?其中,残基Asn39,Gly105和Glu162的位置随机遍布整个结构,其余的残基却全部都明显地聚于一个靠近铁原子的子类当中?由此从在三维结构确认sod基因编码含铁的超氧化物歧化酶.此外,那些被识别的靠近铁原子的踪迹残基可能跟铁的绑定和催化功能直接相关?     

C‐terminal tail derived from the neighboring subunit is critical for the activity of Thermoplasma acidophilumD‐aldohexose dehydrogenase

The D ‐aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D ‐aldohexoses, especially D ‐mannose. AldT comprises a unique C‐terminal tail motif (residues 247–255) that shuts the active‐site pocket of the neighboring subunit. The functional role of the C‐terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C‐terminal deletion mutants (Δ254, Δ253, Δ252, and Δ249) and two site‐specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C‐terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Δ249 mutant was also determined. The structure showed that the active‐site loops undergo a significant conformational change, which leads to the structural deformation of the substrate‐binding pocket. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Structural insights into unique substrate selectivity of Thermoplasma acidophilum D-aldohexose dehydrogenase

The d-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD⁺ rather than NADP⁺ as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against d-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate d-mannose, at 2.1 Å, 1.65 Å, and 1.6 Å resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The d-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound d-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between d-mannose and its C2 epimer d-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the d-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.     

Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high‐pH RP separation with conventional low‐pH RP separation in a two‐dimensional capillary liquid chromatography (2‐D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first‐dimension, high‐pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second‐dimension, low‐pH RP separation, each under identical gradient‐elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non‐redundant proteins, of which 50% were observed in all three replicates. A comparison of RP‐RP 2‐D LC and strong cation exchange‐RP 2‐D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.     

Improving basic and membrane protein MS detection of the culture filtrate proteins from Mycobacterium tuberculosis H37Rv by biomimetic affinity prefractionation

The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines, and diagnostics. The constituents of the M. tuberculosis CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel‐based prefractionation is usually performed before MS analysis. In this study, we describe a novel prefractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six‐column cascade. The LC‐MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first‐time identifications. Notably, ～1/3 (20/61) of these novel CFPs are membrane proteins, among which nine proteins have 2–14 transmembrane domains. In addition, ～1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that biomimetic affinity chromatography prefractionation markedly improves protein detection by LC‐MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased.     

Protein interactomics based on direct molecular fishing on paramagnetic particles: Practical realization and further SPR validation

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity‐based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC?MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR‐based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 μM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein–protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.     

Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.     

Isoforms of wild type proteins often appear as low molecular weight bands on SDS‐PAGE

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS‐PAGE), is a technique commonly used to detect specific proteins. SDS‐PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography – mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non‐malignant HEK293 and cancerous MDA‐MB231 (MB231) cells separated using SDS‐PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS‐PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer‐therapy studies.     

Identification of lysosomal Npc1‐binding proteins: Cathepsin D activity is regulated by NPC1

Niemann–Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1‐binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032–1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC‐MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.     

Shedding & shaving: disclosure of proteomic expressions on a bacterial face

Bacterial surface proteins are critically important in determining the success of bacterial strains in their competition for survival, which makes comprehensive knowledge of the microbial 'face' of high relevance to understand bacterial physiology. Therefore, the aim of this study was to inventorize the proteomic composition of a bacterial surface by gel-free approaches using Bacillus subtilis as a model organism. To this purpose, intact bacteria were incubated with trypsin to cleave surface proteins, after which these 'shaved' proteins were identified by LC-MS/MS analysis of tryptic peptides that were released into the incubation buffer. In parallel, proteins that were released from bacterial cells without trypsin treatment were inventorized and defined as 'shed' proteins. The results showed that peptides of 41 proteins, comprising transmembrane proteins, secretory and lipoproteins, known 'anchorless' surface proteins and ribosomal proteins, were specifically released by shaving. Some of these proteins were also cleaved by trypsin-beads, which are unable to penetrate the bacterial cell wall, indicating a genuine surface-exposed localization. Together this shows that these combined gel-free approaches can reveal novel proteomic decorations on a bacterial face and may provide leads for future studies on protein sorting in B. subtilis and other Gram-positive bacteria.     

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.     

Proteomic analysis of the tetraspanin web using LC-ESI-MS/MS and MALDI-FTICR-MS

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of a molecular network of interactions, the "tetraspanin web". Here, we have performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of two cell lines derived from primary tumor and metastasis from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification and the proteins were identified by MS using LC-ESI-MS/MS and MALDI-FTICR. The high resolution and mass accuracy of FTICR MS allowed reliable identification using mass finger printing with only two peptides. Thus, it could be used to resolve the composition of complex peptide mixtures from membrane proteins. Different types of membrane proteins were identified, including adhesion molecules (integrins, Lu/B-CAM, GA733 proteins), receptors and signaling molecules (BAI2, PKC, G proteins), proteases (ADAM10, TADG15), and membrane fusion proteins (syntaxins) as well as poorly characterized proteins (CDCP1, HEM-1, CTL1, and CTL2). Some components were differentially detected in the tetraspanin web of the two cell lines. These differences may be relevant for tumor progression and metastasis.