Proteome Analysis of the Surface of Trichomonas vaginalis Reveals Novel Proteins and Strain-dependent Differential Expression

The identification of surface proteins on the plasma membrane of pathogens is of fundamental importance in understanding host-pathogen interactions. Surface proteins of the extracellular parasite Trichomonas are implicated in the initial adherence to mucosal tissue and are likely to play a critical role in the long term survival of this pathogen in the urogenital tract. In this study, we used cell surface biotinylation and multidimensional protein identification technology to identify the surface proteome of six strains of Trichomonas vaginalis with differing adherence capacities to vaginal epithelial cells. A combined total of 411 proteins were identified, and of these, 11 were found to be more abundant in adherent strains relative to less adherent parasites. The mRNA levels of five differentially expressed proteins selected for quantitative RT-PCR analysis mirrored their observed protein levels, confirming their up-regulation in highly adherent strains. As proof of principle and to investigate a possible role in pathogenesis for differentially expressed proteins, gain of function experiments were performed using two novel proteins that were among the most highly expressed surface proteins in adherent strains. Overexpression of either of these proteins, TVAG_244130 or TVAG_166850, in a relatively non-adherent strain increased attachment of transfected parasites to vaginal epithelial cells ∼2.2-fold. These data support a role in adhesion for these abundant surface proteins. Our analyses demonstrate that comprehensive profiling of the cell surface proteome of different parasite strains is an effective approach to identify potential new adhesion factors as well as other surface molecules that may participate in establishing and maintaining infection by this extracellular pathogen.The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide with an estimated 174 million new cases annually (1). Although asymptomatic infection by T. vaginalis is common, multiple symptoms and pathologies can arise in both men and women, including vaginitis, urethritis, prostatitis, low birth weight infants and preterm delivery, premature rupture of membranes, and infertility (2–5). T. vaginalis has also emerged as an important cofactor in amplifying human immunodeficiency virus spread (6) as individuals infected with T. vaginalis have a significantly increased incidence of human immunodeficiency virus transmission (7, 8). T. vaginalis infection likewise increases the risk of cervical and aggressive prostate cancers (9–11).Despite the serious consequences that can arise from trichomoniasis, the underlying biochemical processes that lead to T. vaginalis pathogenesis are not well defined. Because T. vaginalis is an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host (12). Several in vitro studies indicate that adhesion of the parasite to target mucosal epithelial cells is essential for the maintenance of infection and for cytopathogenicity (13, 14). T. vaginalis adherence to host cells is mediated, in part, by a lipophosphoglycan (LPG)¹ that coats the surface of the parasite, and altering the sugar content of this LPG reduces both adherence and cytotoxicity (15). Moreover, the mammalian protein galectin-1 binds to T. vaginalis in a carbohydrate-dependent manner via a direct interaction with parasite LPG (16). Knockdown of galectin-1 in mammalian cells, however, reduces parasite binding only by ∼17% (16). Although galectin-1-mediated interactions between T. vaginalis LPG and host cell glycoconjugates may be central in establishing infection, it is clear that parasite adhesion factors in addition to LPG are likely to be involved in host-parasite interaction. Surface proteins are likely to play important roles in the initial adherence to mucosal tissue as well as the long term survival of the pathogen on mucosal surfaces.The outcome of infection with T. vaginalis is highly variable. Possible explanations for this phenomenon include host immunity, host nutritional status, and the vaginal microbiota. Additionally, genetic differences between T. vaginalis isolates leading to differences in adherence and cytotoxicity capacities are likely to result in differences in disease progression. Recently, geographically diverse T. vaginalis strains that are significantly more cytotoxic to host cells than laboratory-adapted strains have become available (17, 18), paving the way toward comparative studies aimed at identifying proteins that correlate with virulent phenotypes.Despite the importance of T. vaginalis surface proteins as a critical interface for pathogen-host interactions, there has been no systematic investigation of the surface proteins of this parasite. The T. vaginalis genome is large and encodes a massive proteome with a considerable and diverse repertoire of candidate surface proteins (19). For example, sequence analysis programs that predict transmembrane protein topology identified over 5100 T. vaginalis proteins with one or more transmembrane domains (20). Furthermore, over 300 annotated proteins with predicted transmembrane domains also contain protein motifs common to surface proteins from other pathogens known to contribute to mucosal colonization and other pathogenic processes (20). The vast number and diversity of possible surface proteins necessitates a multitiered approach using complementary genomics and proteomics analyses to identify candidates for focused functional studies.Biotinylation of proteins at the cell surface with an impermeable reagent followed by specific purification of these proteins using streptavidin has successfully been used for the enrichment and identification of surface proteins (21–24). The high avidity binding of biotin to streptavidin greatly enhances membrane protein purification, a challenging feat because of the low abundance of membrane proteins in total cellular extracts. Here, we used this approach to profile the surface plasma membrane proteome of T. vaginalis and to identify proteins that are differentially expressed in adherent relative to less adherent strains of the parasite. To the best of our knowledge, this is the first study to systematically identify and characterize proteins at the surface of Trichomonas parasites. Defining the parasite cell surface proteome is a critical step toward understanding the relative abundance of surface proteins in strains with varying virulence properties. This information will be critical for defining the role surface proteins play in mediating contact between the parasite and host cells as well as the resulting intracellular and extracellular signals that contribute to establishing and maintaining infection. Additionally, conserved surface molecules unique to T. vaginalis that might serve as specific vaccine candidates can be revealed using this approach. The prevalence of trichomoniasis among women of reproductive age (25) and its correlation with AIDS transmission and cervical and prostate cancers (6, 8–11) provide strong arguments for the need to develop vaccines against this human pathogen.     

Nonparametric Analysis of Thermal Proteome Profiles Reveals Novel Drug-binding Proteins*

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Highlights

• •Method for the analysis of response curves from thermal proteome profiling (TPP).
• •NPARC uses nonparametric statistics and provides false discovery-rate (FDR) control.
• •Increased proteome coverage and sensitivity to identify drug-binding proteins.

     

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter‐individual variability reported, confident, and reliable insights have been hindered. Using label‐free quantitative (LFQ)‐proteomics analysis, a reproducible, quantifiable investigation of the 1U mL^?1 thrombin‐induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ‐proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet‐derived exosomes. Vesicles in the exosomal‐size range were confirmed in healthy‐human PR and reduced numbers of similar‐sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha‐granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low‐population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet‐related disease.     

Salivary Gland Proteome Analysis Reveals Modulation of Anopheline Unique Proteins in Insensitive Acetylcholinesterase Resistant Anopheles gambiae Mosquitoes

Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace) encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1^R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1^R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.     

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.     

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells’ antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.     

Serum Proteome and Cytokine Analysis in a Longitudinal Cohort of Adults with Primary Dengue Infection Reveals Predictive Markers of DHF

Background

Infections caused by dengue virus are a major cause of morbidity and mortality in tropical and subtropical regions of the world. Factors that control transition from mild forms of disease such as dengue fever (DF) to more life-threatening forms such as dengue hemorrhagic fever (DHF) are poorly understood. Consequently, there are no reliable methods currently available for early triage of DHF patients resulting in significant over-hospitalization.

Methodology/Principal Findings

We have systematically examined the proteome, cytokines and inflammatory markers in sera from 62 adult dengue patients (44 DF; 18 DHF) with primary DENV infection, at three different times of infection representing the early febrile, defervescence and convalescent stages. Using fluorescent bioplex assays, we measured 27 cytokines in these serum samples. Additionally, we used multiple mass spectrometry methods for iTRAQ-based comparative analysis of serum proteome as well as measurements of protein adducts- 3-nitrotyrosine and 3-chlorotyrosine as surrogate measures of free radical activity. Using multiple methods such as OPLS, MRMR and MSVM-RFE for multivariate feature selection and classification, we report molecular markers that allow prediction of primary DHF with sensitivity and specificity of >80%.

Conclusions/Significance

This report constitutes a comprehensive analysis of molecular signatures of dengue disease progression and will help unravel mechanisms of dengue disease progression. Our analysis resulted in the identification of markers that may be useful for early prediction of DHF during the febrile phase. The combination of highly sensitive analytical methods and novel statistical approaches described here forms a robust platform for biomarker discovery.     

Identification of Chlamydia trachomatis Outer Membrane Complex Proteins by Differential Proteomics

The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.Bacteria in the phylum Chlamydiae are characterized by their complex intracellular developmental cycles. Chlamydiae must assume at least two functionally distinct morphotypes, the intracellular, replicative reticulate body (RB) and the extracellular, infectious elementary body (EB), to replicate and be transmitted to new hosts (50). The divergence of distinct RB and EB forms may have been driven by the different pressures these pathogens face inside host cells during replication and outside host cells during transmission. For example, the outer membrane of EB contains a poorly immunogenic truncated lipopolysaccharide (LPS) (14, 30) and immunodominant epitopes of the major outer membrane protein (MOMP) vary substantially among closely related chlamydial strains (13). EB also lack detectable peptidoglycan (2, 20, 60), although functional murein biosynthetic enzymes (2, 5, 16, 21, 32, 43, 45, 46) are expressed in RB during productive and persistent infection (44). To compensate for the loss of murein, EB are enveloped by a protein P-layer, which lends osmotic stability to the infectious particle (29).Attempts to identify components of the P-layer and outer membrane proteins of Chlamydia were advanced by the observation that these layers can be separated from many soluble EB proteins using the detergent N-lauroyl sarcosine (Sarkosyl). Caldwell et al. dubbed the Sarkosyl-insoluble fraction the chlamydial outer membrane complex (COMC) and noted that purified COMC maintained the shape of intact EB and contained a complete outer membrane, and they reported that a single outer membrane protein, MOMP, accounted for more than 60% of total COMC protein content (15). Other studies revealed that the COMC is stabilized by extensive disulfide bonds between MOMP monomers (26, 27, 53) and between MOMP and two abundant cysteine-rich COMC components (26, 28). Other studies revealed that the COMC is stabilized by extensive disulfide bonds between MOMP monomers (18, 29, 52) and the EB surface (3, 19, 47, 67). More recent data suggest that not all EB outer membrane (OM) proteins are disulfide cross-linked to the COMC. For example, polymorphic membrane protein D localizes to the surface of EB but can be extracted from intact EB with gentle detergents in the absence of reducing agents (17, 61). Thus, not all COMC proteins are exposed on the EB surface, nor are all EB OM proteins components of the COMC.Beyond these well-described and abundant COMC components, other studies have indicated that additional proteins localize to the EB surface and/or COMC of Chlamydia trachomatis (7, 28, 36, 51, 57, 64, 67, 70). However, confirming that specific proteins localize to the COMC or OM of EB can be challenging due to factors such as the contamination of EB preparations with RB proteins and technical limitations of proteomic and surface-labeling protein identification methods (29, 56).Here, we used differential proteomics to identify proteins specifically enriched in the COMC. Isolated COMC were dissolved in 8 M urea, and the extracted proteins were digested with trypsin. The resulting peptides were analyzed by high-sensitivity liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to identify low-abundance proteins. Sarkosyl-soluble fractions and whole EB were analyzed in parallel with COMC, and protein assignments were compared among three replicate runs of each fraction. In total, peptides from 329 L2 proteins were identified. The differential analysis of protein abundance indicated the enrichment of 17 proteins in the COMC. Our results define the cadre of low-abundance COMC proteins, provide a starting point for the identification of surface-exposed EB proteins, and identify EB proteins that are likely to be recognized by innate immunity receptors and/or capable of eliciting neutralizing antibodies in vivo. Finally, our findings and data from other recent studies permit the refinement of existing models of EB and COMC structure.     

Directed Evolution Reveals Hidden Properties of VMAT, a Neurotransmitter Transporter

The vesicular neurotransmitter transporter VMAT2 is responsible for the transport of monoamines into synaptic and storage vesicles. VMAT2 is the target of many psychoactive drugs and is essential for proper neurotransmission and survival. Here we describe a new expression system in Saccharomyces cerevisiae that takes advantage of the polyspecificity of VMAT2. Expression of rVMAT2 confers resistance to acriflavine and to the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP⁺) by their removal into the yeast vacuole. This expression system allowed identification of a new substrate, acriflavine, and isolation of mutants with modified affinity to tetrabenazine (TBZ), a non-competitive inhibitor of VMAT2 that is used in the treatment of various movement disorders including Tourette syndrome and Huntington chorea. Whereas one type of mutant obtained displayed decreased affinity to TBZ, a second type showed only a slight decrease in the affinity to TBZ, displayed a higher K_m to the neurotransmitter serotonin, but conferred increased resistance to acriflavine and MPP⁺. A protein where both types of mutations were combined (with only three amino acid replacements) lost most of the properties of the neurotransmitter transporter (TBZ-insensitive, no transport of neurotransmitter) but displayed enhanced resistance to the above toxicants. The work described here shows that in the case of rVMAT2, loss of traits acquired in evolution of function (such as serotonin transport and TBZ binding) bring about an improvement in older functions such as resistance to toxic compounds. A process that has taken millions of years of evolution can be reversed by three mutations.     

Proteome Analysis of Plasmodium falciparum Extracellular Secretory Antigens at Asexual Blood Stages Reveals a Cohort of Proteins with Possible Roles in Immune Modulation and Signaling

The highly co-evolved relationship of parasites and their hosts appears to include modulation of host immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood. Characterization of these key genes and their cognate proteins related to the host-parasite interplay should lead to a better understanding of this intriguing biological phenomenon. The malaria agent Plasmodium falciparum is predicted to export a cohort of several hundred proteins to remodel the host erythrocyte. However, proteins actively exported by the asexual intracellular parasite beyond the host red blood cell membrane (before merozoite egress) have been poorly investigated so far. Here we used two complementary methodologies, two-dimensional gel electrophoresis/MS and LC-MS/MS, to examine the extracellular secreted antigens at asexual blood stages of P. falciparum. We identified 27 novel antigens exported by P. falciparum in the culture medium of which some showed clustering with highly polymorphic genes on chromosomes, suggesting that they may encode putative antigenic determinants of the parasite. Immunolocalization of four novel secreted proteins confirmed their export beyond the infected red blood cell membrane. Of these, preliminary functional characterization of two novel (Sel1 repeat-containing) parasite proteins, PfSEL1 and PfSEL2 revealed that they down-regulate expression of cell surface Notch signaling molecules in host cells. Also a novel protein kinase (PfEK) and a novel protein phosphatase (PfEP) were found to, respectively, phosphorylate/dephosphorylate parasite-specific proteins in the extracellular culture supernatant. Our study thus sheds new light on malaria parasite extracellular secreted antigens of which some may be essential for parasite development and could constitute promising new drug targets.Plasmodium falciparum is a wide spread protozoan parasite responsible for over a million deaths annually mainly among children in sub-Saharan Africa (1). Like other apicomplexan parasites such as Leishmania, Trypanosoma, and Toxoplasma, Plasmodia depend on a series of intricate and highly evolved adaptations that enable them to evade destruction by the host immune responses. These protozoan parasites have provided some of the best leads in elucidating the mechanisms to circumvent innate immunity and adaptive humoral and cellular immunity (2). Ingenious strategies to escape innate defenses include subversion of attack by humoral effector mechanisms such as complement lysis and lysis by other serum components (3), remodeling of phagosomal compartments in which they reside (4), modulation of host cell signaling pathways (5), and modification of the antigen-presenting and immunoregulatory functions of dendritic cells, which provide a crucial link with the adaptive immune response (6). Malaria parasites also predominantly use antigenic diversity and clonal antigenic variation to evade adaptive immunity of the host (7). Surface-associated and secreted parasite proteins are major players in host-parasite cross-talk and are advantageously used by the parasite to counter the host immune system. Proteins secreted by a wide range of parasitic pathogens into the host microenvironment result in symptomatic infections. For example, the excretory-secretory (ES)¹ products of the parasitic fluke Fasciola hepatica are key players in host-parasite interactions (8). Among the apicomplexans, proteomics analyses of rhoptry organelles of Toxoplasma gondii have revealed many novel constituents of host-parasite interactions (9).The identification and trafficking of Plasmodium proteins exported into the host erythrocyte have been subjects of recent detailed investigations. A number of studies have identified Plasmodium proteins that contain signature sequence motifs, the host cell targeting signal or the Plasmodium export element (PEXEL), that target these proteins into the infected erythrocytes (10, 11). Recent proteomics analyses have identified novel proteins in the raftlike membranes of the parasite and on the surface of infected erythrocytes (12, 13). P. falciparum translationally controlled tumor protein (PfTCTP), a homolog of the mammalian histamine-releasing factor, has been shown to be released into the culture supernatant from intact as well as ruptured infected RBCs and causes histamine release from human basophils and IL-8 secretion from eosinophils (14). However, the total spectrum of proteins actively exported by the asexual intracellular parasite beyond the host RBC membrane (before merozoite egress) has been poorly investigated so far.In the present study, we used two complementary methodologies, two-dimensional gel electrophoresis (2DE)/MS and LC-MS/MS to examine the cohort of extracellular secreted antigens (ESAs) at asexual blood stages of P. falciparum. Our findings reveal that malaria parasites secrete a number of effector molecules such as immunomodulators and signaling proteins that are potentially involved in host-parasite interactions. Prominent among these are proteins with Sel1 domain, a protein of the LCCL family, a novel protein kinase, and a novel protein phosphatase. Secreted-extracellular/iRBC surface localization of some of these proteins was validated by immunolocalization studies. We also characterized the functions of some of these proteins in the culture supernatant, thus providing an insight into the nature of some of the malaria parasite extracellular antigens.     

Rare Dissipative Transitions Punctuate the Initiation of Chemical Denaturation in Proteins

Protein unfolding dynamics are bound by their degree of entropy production, a quantity that relates the amount of heat dissipated by a nonequilibrium process to a system’s forward and time-reversed trajectories. We here explore the statistics of heat dissipation that emerge in protein molecules subjected to a chemical denaturant. Coupling large molecular dynamics datasets and Markov state models with the theory of entropy production, we demonstrate that dissipative processes can be rigorously characterized over the course of the urea-induced unfolding of the protein chymotrypsin inhibitor 2. By enumerating full entropy production probability distributions as a function of time, we first illustrate that distinct passive and dissipative regimes are present in the denaturation dynamics. Within the dissipative dynamical region, we next find that chymotrypsin inhibitor 2 is strongly driven into unfolded states in which the protein’s hydrophobic core has been penetrated by urea molecules and disintegrated. Detailed analyses reveal that urea’s interruption of key hydrophobic contacts between core residues causes many of the protein’s native structural features to dissolve.     

Proteome Analysis of Aniline-Induced Proteins in Acinetobacter lwoffii K24

Acinetobacter lwoffii K24 is a soil bacterium that can use aniline as a sole carbon and nitrogen source (by β-ketoadipate pathway genes (cat genes)) and has two copies of catABC gene separately located on the chromosome. In order to identify aniline-induced proteins, two-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of A. lwoffii K24 cultured in aniline and succinate media. In the range of pH3–10, more than 370 spots were detected on the silver stained gels. Interestingly, more than 20 spots were selectively induced on aniline-cultured bacteria. Twenty-three protein spots of A. lwoffii K24 were analyzed by N-terminal microsequencing and internal microsequencing with in-gel digestion. Of 20 aniline induced protein spots, we identified six β-ketoadipate pathway genes, one subunit of amino group transfer (putative subunit of aniline oxygenase), malate dehydrogenase, putative ABC transporter, putative hydrolase, HHDD isomerase, and five unknown proteins. Especially in case of two catechol 1,2-dioxygenases (CDI₁ and CDI₂), more than three isotypes were detected on the 2D gel. This study showed that the proteome analysis of A. lwoffii K24 may be helpful for identification of genes induced by aniline and understanding of their function in the cell. Received: 2 April 2001 / Accepted: 14 May 2001     

Proteome Profile of American Hybrid Grape cv. Blanc du Bois during Ripening Reveals Proteins Associated with Flavor Volatiles and Ethylene Production

The study of key control points in ripening is essential to improve grape wine quality. Molecular basis of ripening is still far from being understood from the Pierce's disease (PD)‐tolerant grapes predominantly grown in the southeastern United States. To identify proteins expressed during Blanc du Bois grape berry green and ripening stages, proteome analysis from five different stages revealed 1091, 1131, 1078, 1042, and 1066 proteins. Differential expression analysis revealed 551 common proteins across different stages of maturity that are involved in various biochemical and metabolic pathways. The proteins identified were associated with phenylpropanoids, isoquinoline alkaloids, fatty acids, unsaturated fatty acids, and furanones. Our data provide the first step to understand the complex biochemical changes during ripening of PD‐tolerant American hybrid grapes that are popular for their aroma and flavor profile in the southeastern United States. Proteomics data are deposited to the ProteomeXchange PXD004157.