Amino-terminal Signal Peptide Research Articles

Quantitative Mass Spectrometry Characterizes Client Spectra of Components for Targeting of Membrane Proteins to and Their Insertion into the Membrane of the Human ER.

To elucidate the redundancy in the components for the targeting of membrane proteins to the endoplasmic reticulum (ER) and/or their insertion into the ER membrane under physiological conditions, we previously analyzed different human cells by label-free quantitative mass spectrometry. The HeLa and HEK293 cells had been depleted of a certain component by siRNA or CRISPR/Cas9 treatment or were deficient patient fibroblasts and compared to the respective control cells by differential protein abundance analysis. In addition to clients of the SRP and Sec61 complex, we identified membrane protein clients of components of the TRC/GET, SND, and PEX3 pathways for ER targeting, and Sec62, Sec63, TRAM1, and TRAP as putative auxiliary components of the Sec61 complex. Here, a comprehensive evaluation of these previously described differential protein abundance analyses, as well as similar analyses on the Sec61-co-operating EMC and the characteristics of the topogenic sequences of the various membrane protein clients, i.e., the client spectra of the components, are reported. As expected, the analysis characterized membrane protein precursors with cleavable amino-terminal signal peptides or amino-terminal transmembrane helices as predominant clients of SRP, as well as the Sec61 complex, while precursors with more central or even carboxy-terminal ones were found to dominate the client spectra of the SND and TRC/GET pathways for membrane targeting. For membrane protein insertion, the auxiliary Sec61 channel components indeed share the client spectra of the Sec61 complex to a large extent. However, we also detected some unexpected differences, particularly related to EMC, TRAP, and TRAM1. The possible mechanistic implications for membrane protein biogenesis at the human ER are discussed and can be expected to eventually advance our understanding of the mechanisms that are involved in the so-called Sec61-channelopathies, resulting from deficient ER protein import.

Functional characterization of phospholipase B enzyme from Giardia lamblia

The microaerotolarent amitochondriate protozoan Giardia lamblia causes Giardiasis and produces a unique enzyme called Phospholipase B (PLB) in contrast to higher eukaryotes. The enzyme is produced upon induction with oxidative (H2O2) stress, thus leading to prostaglandin E2 (PGE2) production. It exists in dimeric form, and its molecular weight is 56 kDa. This PLB was extracellularly cloned in the pET21d vector. The ORF is 1620 bp (Genbank accession no. -OM939681) long and codes for a protein 539 amino acid long, with a 15 amino acid long amino-terminal signal peptide. The highest enzyme activity of PLB was identified at pH 7.5 and 35 °C. This specific enzyme was also active at 50 °C pH 10, but activity was low. We also analyzed the expression of PLB protein in G. lamblia, which was significantly induced under increased oxidative stress.

Signal Peptide Features Determining the Substrate Specificities of Targeting and Translocation Components in Human ER Protein Import.

In human cells, approximately 30% of all polypeptides enter the secretory pathway at the level of the endoplasmic reticulum (ER). This process involves cleavable amino-terminal signal peptides (SPs) or more or less amino-terminal transmembrane helices (TMHs), which serve as targeting determinants, at the level of the precursor polypeptides and a multitude of cytosolic and ER proteins, which facilitate their ER import. Alone or in combination SPs and TMHs guarantee the initial ER targeting as well as the subsequent membrane integration or translocation. Cytosolic SRP and SR, its receptor in the ER membrane, mediate cotranslational targeting of most nascent precursor polypeptide chains to the polypeptide-conducting Sec61 complex in the ER membrane. Alternatively, fully-synthesized precursor polypeptides and certain nascent precursor polypeptides are targeted to the ER membrane by either the PEX-, SND-, or TRC-pathway. Although these targeting pathways may have overlapping functions, the question arises how relevant this is under cellular conditions and which features of SPs and precursor polypeptides determine preference for a certain pathway. Irrespective of their targeting pathway(s), most precursor polypeptides are integrated into or translocated across the ER membrane via the Sec61 channel. For some precursor polypeptides specific Sec61 interaction partners have to support the gating of the channel to the open state, again raising the question why and when this is the case. Recent progress shed light on the client spectrum and specificities of some auxiliary components, including Sec62/Sec63, TRAM1 protein, and TRAP. To address the question which precursors use a certain pathway or component in intact human cells, i.e., under conditions of fast translation rates and molecular crowding, in the presence of competing precursors, different targeting organelles, and relevant stoichiometries of the involved components, siRNA-mediated depletion of single targeting or transport components in HeLa cells was combined with label-free quantitative proteomics and differential protein abundance analysis. Here, we present a summary of the experimental approach as well as the resulting differential protein abundance analyses and discuss their mechanistic implications in light of the available structural data.

Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing.

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

Epidermal Growth Factor-Like Domain-8 (EGFL8) in Mammals: The Story so far


 
 
 Epidermal growth factor-like domain-8 (EGFL8) also known as vascular endothelial statin-2 (VE-statin-2). It was identified by using retroviral gene entrapment vectors, expressed in endothelial cells. It is located on chromosome6inhumansandchromosome17inthemouse. EGFL8codesaproteinof293aminoacidswithan amino-terminal signal peptide and has two EGF-like domains. EGFL8 plays a very important regulatory role in thymopoiesis, cell migration and invasion through the modulation of Notch signaling. Although the signaling regulatory factors of EGFL8 need to be explored but recent scientific advances have revealed some important aspect of its regulation. This review summarizes the current knowledge about all aspects of EGFL8 since its discovery.
 
 

TRAM1 protein may support ER protein import by modulating the phospholipid bilayer near the lateral gate of the Sec61-channel

ABSTRACT In mammalian cells, one-third of all polypeptides is transported into or through the ER-membrane via the Sec61-channel. While the Sec61-complex facilitates the transport of all polypeptides with amino-terminal signal peptides (SP) or SP-equivalent transmembrane helices (TMH), the translocating chain-associated membrane protein (now termed TRAM1) was proposed to support transport of a subset of precursors. To identify possible determinants of TRAM1 substrate specificity, we systematically identified TRAM1-dependent precursors by analyzing cellular protein abundance changes upon TRAM1 depletion in HeLa cells using quantitative label-free proteomics. In contrast to previous analysis after TRAP depletion, SP and TMH analysis of TRAM1 clients did not reveal any distinguishing features that could explain its putative substrate specificity. To further address the TRAM1 mechanism, live-cell calcium imaging was carried out after TRAM1 depletion in HeLa cells. In additional contrast to previous analysis after TRAP depletion, TRAM1 depletion did not affect calcium leakage from the ER. Thus, TRAM1 does not appear to act as SP- or TMH-receptor on the ER-membrane’s cytosolic face and does not appear to affect the open probability of the Sec61-channel. It may rather play a supportive role in protein transport, such as making the phospholipid bilayer conducive for accepting SP and TMH in the vicinity of the lateral gate of the Sec61-channel. Abbreviations: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; RAMP, ribosome-associated membrane protein; SP, signal peptide; SR, SRP-receptor; SRP, signal recognition particle; TMH, signal peptide-equivalent transmembrane helix; TRAM, translocating chain-associated membrane protein; TRAP, translocon-associated protein.

Export of a Vibrio parahaemolyticus toxin by the Sec and type III secretion machineries in tandem.

Many Gram-negative pathogens utilize dedicated secretion systems to export virulence factors such as exotoxins and effectors1-4. Several exotoxins are synthesized as precursors containing amino-terminal Sec signal peptides and are exported through the inner-membrane-bound Sec machinery to the periplasm, followed by secretion across the outer membrane to the exterior using a type II secretion system (T2SS)3,5. Here, we report that thermostable direct haemolysin (TDH), an exotoxin of the food-borne pathogen Vibrio parahaemolyticus, can be exported through the type III secretion system (T3SS), which engages in one-step secretion of effectors4, despite possessing a Sec signal peptide and being mainly secreted via the T2SS. Although the precursor of TDH is targeted to the Sec pathway, a fraction of mature TDH was observed to re-enter the bacterial cytoplasm. The N terminus of mature TDH comprises a T3SS signal sequence, allowing it to be loaded into the T3SS. We also show that T3SS-delivered TDH as an effector contributes to intestinal fluid accumulation in a rabbit diarrhoeal model of V. parahaemolyticus infection. Thus, our results show that an unconventional export mechanism for a bacterial toxin via the T3SS in tandem with the Sec machinery facilitates the virulence trait of V. parahaemolyticus.

Proteomics reveals signal peptide features determining the client specificity in human TRAP-dependent ER protein import

In mammalian cells, one-third of all polypeptides are transported into or across the ER membrane via the Sec61 channel. While the Sec61 complex facilitates translocation of all polypeptides with amino-terminal signal peptides (SP) or transmembrane helices, the Sec61-auxiliary translocon-associated protein (TRAP) complex supports translocation of only a subset of precursors. To characterize determinants of TRAP substrate specificity, we here systematically identify TRAP-dependent precursors by analyzing cellular protein abundance changes upon TRAP depletion using quantitative label-free proteomics. The results are validated in independent experiments by western blotting, quantitative RT-PCR, and complementation analysis. The SPs of TRAP clients exhibit above-average glycine-plus-proline content and below-average hydrophobicity as distinguishing features. Thus, TRAP may act as SP receptor on the ER membrane’s cytosolic face, recognizing precursor polypeptides with SPs of high glycine-plus-proline content and/or low hydrophobicity, and triggering substrate-specific opening of the Sec61 channel through interactions with the ER-lumenal hinge of Sec61α.

Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans

BackgroundThe gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces.ResultsCelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498–507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50–90 mg/L or 100–120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans.ConclusionThis study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.

Identification and Characterization of Daurichromenic Acid Synthase Active in Anti-HIV Biosynthesis.

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells.

Diverse C-Terminal Sequences Involved in Flavobacterium johnsoniae Protein Secretion.

Flavobacteriumjohnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to the family TIGR04183 (type A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign superfolder green fluorescent protein (sfGFP) that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case, approximately 80 to 100 amino acids from the extreme carboxy termini were needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting the secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to the family TIGR04131, which we refer to as a type B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1,182 amino acids to sfGFP failed to result in secretion. Additional features outside the C-terminal region of SprB may be required for its secretion.IMPORTANCE Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to the protein domain family TIGR04183 (type A CTDs) or TIGR04131 (type B CTDs). Here, we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type B CTDs failed to target sfGFP for secretion, suggesting a more complex association with the T9SS.

Protein Targeting to the Plastid of Euglena.

The lateral transfer of photosynthesis between kingdoms through endosymbiosis is among the most spectacular examples of evolutionary innovation. Euglena, which acquired a chloroplast indirectly through an endosymbiosis with a green alga, represents such an example. As with other endosymbiont-derived plastids from eukaryotes, there are additional membranes that surround the organelle, of which Euglena has three. Thus, photosynthetic genes that were transferred from the endosymbiont to the host nucleus and whose proteins are required in the new plastid, are now faced with targeting and plastid import challenges. Early immunoelectron microscopy data suggested that the light-harvesting complexes, photosynthetic proteins in the thylakoid membrane, are post-translationally targeted to the plastid via the Golgi apparatus, an unexpected discovery at the time. Proteins targeted to the Euglena plastid have complex, bipartite presequences that direct them into the endomembrane system, through the Golgi apparatus and ultimately on to the plastid, presumably via transport vesicles. From transcriptome sequencing, dozens of plastid-targeted proteins were identified, leading to the identification of two different presequence structures. Both have an amino terminal signal peptide followed by a transit peptide for plastid import, but only one of the two classes of presequences has a third domain-the stop transfer sequence. This discovery implied two different transport mechanisms; one where the protein was fully inserted into the lumen of the ER and another where the protein remains attached to, but effectively outside, the endomembrane system. In this review, we will discuss the biochemical and bioinformatic evidence for plastid targeting, discuss the evolution of the targeting system, and ultimately provide a working model for the targeting and import of proteins into the plastid of Euglena.

Effect of glucose as a carbon repressor on the extracellular proteome of Aspergillus niger during the production of amylases by solid state cultivation

Abstract In comparison to traditional submerged cultivations, solid state cultivation (SSC) has been shown to reduce the effect of carbon catabolite repression during the production of important industrial enzymes, such as amylases. Nevertheless, the effect of a carbon repressor on the entire secretome during the production of enzymes of industrial interest has not been described. Therefore, the secretome composition and amylase production from Aspergillus niger grown in SSC using media containing starch and different concentrations of glucose (60 and 120 g/L) were evaluated. The results showed that proteins without an amino-terminal signal peptide, which is necessary for secretion through the classical secretory pathway, were detected at high glucose concentrations; some of these proteins were moonlighting proteins associated with pathogenesis. High concentrations of glucose also produced better amylase volumetric activities. α-Amylase activity was eight times higher in media containing glucose as compared to the medium containing only starch. Glucoamylase activity was 29 and 20 times higher at glucose concentrations of 60 and 120 g/L, respectively, compared to the medium containing only starch. Moreover, the presence of glucose had a variable effect on the abundance of the amylase isoforms produced by A. niger.

Characterisation of the secretome of the clam parasite, QPX

Secreted and cell surface-associated molecules play a major role in disease development processes and host-pathogen interactions, and usually determine the virulence of invading organisms. In this study, we investigated proteins secreted by quahog parasite unknown, a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria. In silico analysis of quahog parasite unknown transcripts predicted over 1200 proteins to possess an amino-terminal signal peptide which directs proteins into the classical eukaryotic secretory pathway. Proteomic analysis using LC/MS technology identified 56 proteins present in the extracellular secretion of quahog parasite unknown cells grown in vitro, including six mucin-like molecules, four glycosyl hydrolases and eight peptidases. Transcription levels of 19 quahog parasite unknown extracellular proteins were investigated in clam tissue lesions (in vivo) using quantitative PCR. The overexpression of six of these extracellular proteins in clam tissues compared with in vitro cultures suggests that they are involved in interaction with the clam host.

Unconventional secretion of the mutated 19 kDa protein of Oplophorus luciferase (nanoKAZ) in mammalian cells

The putative amino-terminal signal peptide of the catalytic 19kDa protein (KAZ) of Oplophorus luciferase was found to be a functional secretory peptide in mammalian cells. A 16 amino acid substituted mutant of KAZ (nanoKAZ) could be secreted from mammalian cells using the amino-terminal signal peptide of KAZ, but KAZ could not be secreted at all. Notably, nanoKAZ lacking the amino-terminal signal peptide could be secreted from mammalian cells, and the distribution of nanoKAZ on the cell membrane was confirmed by video-rate bioluminescence imaging. Thus, nanoKAZ lacking the amino-terminal signal peptide was expressed in the cytoplasm, translocated to the cell membrane, and released into the culture medium through an endoplasmic reticulum–Golgi-independent pathway.

Protein transport into the human ER and related diseases, Sec61-channelopathies.

Protein transport into the human endoplasmic reticulum (ER) is relevant to the biogenesis of most soluble and membrane proteins of organelles, which are involved in endo- or exo-cytsosis. It involves amino-terminal signal peptides in the precursor polypeptides and various transport components in the cytosol plus the ER, and can occur co- or post-translationally. The two mechanisms merge at the level of the ER membrane, specifically at the level of the heterotrimeric Sec61 complex, which forms a dynamic polypeptide-conducting channel in the ER membrane. Since the mammalian ER is also the main intracellular calcium storage organelle, and the Sec61 complex is calcium permeable, the Sec61 complex is tightly regulated in its equilibrium between the closed and open conformations, or "gated", by ligands, such as signal peptides of the transport substrates and the ER lumenal Hsp70-type molecular chaperone BiP. Furthermore, BiP binding to the incoming polypeptide contributes to the efficiency and unidirectionality of transport. Recent insights into the structure and dynamic equilibrium of the Sec61 complex have various mechanistic as well as medical implications.

Sub-cellular localization of PELPK1 in Arabidopsis thaliana as determined by translational fusion with green fluorescent protein reporter

PELPK1, a novel Arabidopsis thaliana gene was earlier annotated to encode a protein of sub-family, PELPK under hydroxyproline-rich glycoprotein (HRGP) super-family of proteins. Previous bioinformatics and computational analyses predicted PELPK1 to contain an amino-terminal signal peptide destined towards the secretory pathway. In the present study, transgenic plants were developed harboring a translational fusion construct comprising of PELPK1 coding sequence (PELPK1-CDS) and green fluorescent protein (GFP) reporter to determine the localization of PELPK1 in Arabidopsis plants. By employing the techniques of confocal laser scanning microscopy, immunolabeling of GFP with quantum dot (Q-dot), and transmission electron microscopy (TEM), it is shown that the translational fusion product is predominantly deposited to the cell wall. These results are in agreement with the earlier bioinformatics prediction that the PELPK1 is transported via the secretory pathway.

Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.

Mapping of the SecA Signal Peptide Binding Site and Dimeric Interface by Using the Substituted Cysteine Accessibility Method

SecA is an ATPase nanomotor critical for bacterial secretory protein translocation. Secretory proteins carry an amino-terminal signal peptide that is recognized and bound by SecA followed by its transfer across the SecYEG translocon. While this process is crucial for the onset of translocation, exactly where the signal peptide interacts with SecA is unclear. SecA protomers also interact among themselves to form dimers in solution, yet the oligomeric interface and the residues involved in dimerization are unknown. To address these issues, we utilized the substituted cysteine accessibility method (SCAM); we generated a library of 23 monocysteine SecA mutants and probed for the accessibility of each mutant cysteine to maleimide-(polyethylene glycol)2-biotin (MPB), a sulfhydryl-labeling reagent, both in the presence and absence of a signal peptide. Dramatic differences in MPB labeling were observed, with a select few mutants located at the preprotein cross-linking domain (PPXD), the helical wing domain (HWD), and the helical scaffold domain (HSD), indicating that the signal peptide binds at the groove formed between these three domains. The exposure of this binding site is varied under different conditions and could therefore provide an ideal mechanism for preprotein transfer into the translocon. We also identified residues G793, A795, K797, and D798 located at the two-helix finger of the HSD to be involved in dimerization. Adenosine-5'-(γ-thio)-triphosphate (ATPγS) alone and, more extensively, in conjunction with lipids and signal peptides strongly favored dimer dissociation, while ADP supports dimerization. This study provides key insight into the structure-function relationships of SecA preprotein binding and dimer dissociation.

Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry

Tuberculosis (TB) is an infectious bacterial disease that causes morbidity and mortality, especially in developing countries. Although its efficacy against TB has displayed a high degree of variability (0%-80%) in different trials, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important weapon for preventing TB worldwide for over 80 years. Because secreted proteins often play vital roles in the interaction between bacteria and host cells, the secretome of mycobacteria is considered to be an attractive reservoir of potential candidate antigens for the development of novel vaccines and diagnostic reagents. In this study, we performed a proteomic analysis of BCG culture filtrate proteins using SDS-PAGE and high-resolution Fourier transform mass spectrometry. In total, 239 proteins (1555 unique peptides) were identified, including 185 secreted proteins or lipoproteins. Furthermore, 17 novel protein products not annotated in the BCG database were detected and validated by means of RT-PCR at the transcriptional level. Additionally, the translational start sites of 52 proteins were confirmed, and 22 proteins were validated through extension of the translational start sites based on N-terminus-derived peptides. There are 103 secreted proteins that have not been reported in previous studies on BCG [corrected] secretome and are unique to our study. The physicochemical characteristics of the secreted proteins were determined. Major components from the culture supernatant, including low-molecular-weight antigens, lipoproteins, Pro-Glu and Pro-Pro-Glu family proteins, and Mce family proteins, are discussed; some components represent potential predominant antigens in the humoral and cellular immune responses.