Apex Peptide Elution Chain Selection: A New Strategy for Selecting Precursors in 2D-LC-MALDI-TOF/TOF Experiments on Complex Biological Samples

LC-MALDI provides an often overlooked opportunity to exploit the separation between LC-MS and MS/MS stages of a 2D-LC-MS-based proteomics experiment, that is, by making a smarter selection for precursor fragmentation. Apex Peptide Elution Chain Selection (APECS) is a simple and powerful method for intensity-based peptide selection in a complex sample separated by 2D-LC, using a MALDI-TOF/TOF instrument. It removes the peptide redundancy present in the adjacent first-dimension (typically strong cation exchange, SCX) fractions by constructing peptide elution profiles that link the precursor ions of the same peptide across SCX fractions. Subsequently, the precursor ion most likely to fragment successfully in a given profile is selected for fragmentation analysis, selecting on precursor intensity and absence of adjacent ions that may cofragment. To make the method independent of experiment-specific tolerance criteria, we introduce the concept of the branching factor, which measures the likelihood of false clustering of precursor ions based on past experiments. By validation with a complex proteome sample of Arabidopsis thaliana, APECS identified an equivalent number of peptides as a conventional data-dependent acquisition method but with a 35% smaller work load. Consequently, reduced sample depletion allowed further selection of lower signal-to-noise ratio precursor ions, leading to a larger number of identified unique peptides.

Regulation of ykrL (htpX) by Rok and YkrK, a Novel Type of Regulator in Bacillus subtilis

Expression of ykrL of Bacillus subtilis, encoding a close homologue of the Escherichia coli membrane protein quality control protease HtpX, was shown to be upregulated under membrane protein overproduction stress. Using DNA affinity chromatography, two proteins were found to bind to the promoter region of ykrL: Rok, known as a repressor of competence and genes for extracytoplasmic functions, and YkrK, a novel type of regulator encoded by the gene adjacent to ykrL but divergently transcribed. Electrophoretic mobility shift assays showed Rok and YkrK binding to the ykrL promoter region as well as YkrK binding to the ykrK promoter region. Comparative bioinformatic analysis of the ykrL promoter regions in related Bacillus species revealed a consensus motif, which was demonstrated to be the binding site of YkrK. Deletion of rok and ykrK in a PykrL-gfp reporter strain showed that both proteins are repressors of ykrL expression. In addition, conditions which activated PykrL (membrane protein overproduction, dissipation of the membrane potential, and salt and phenol stress) point to the involvement of YkrL in membrane protein quality control

A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers

Interactions between hematopoietic stem cells and their niche are mediated by proteins within the plasma membrane (PM) and changes in these interactions might alter hematopoietic stem cell fate and ultimately result in acute myeloid leukemia (AML). Here, using nano-LC/MS/MS, we set out to analyze the PM profile of two leukemia patient samples. We identified 867 and 610 unique CD34(+) PM (-associated) proteins in these AML samples respectively, including previously described proteins such as CD47, CD44, CD135, CD96, and ITGA5, but also novel ones like CD82, CD97, CD99, PTH2R, ESAM, MET, and ITGA6. Further validation by flow cytometry and functional studies indicated that long-term self-renewing leukemic stem cells reside within the CD34(+)/ITGA6(+) fraction, at least in a subset of AML cases. Furthermore, we combined proteomics with transcriptomics approaches using a large panel of AML CD34(+) (n = 60) and normal bone marrow CD34(+) (n = 40) samples. Thus, we identified eight subgroups of AML patients based on their specific PM expression profile. GSEA analysis revealed that these eight subgroups are enriched for specific cellular processes

Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H2L2 tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ~392 residues and two L subunits of ~150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ~100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

A Gα-Stimulated RapGEF Is a Receptor-Proximal Regulator of Dictyostelium Chemotaxis

Chemotaxis, or directional movement toward extracellular chemical gradients, is an important property of cells that is mediated through G-protein-coupled receptors (GPCRs). Although many chemotaxis pathways downstream of Gβγ have been identified, few Gα effectors are known. Gα effectors are of particular importance because they allow the cell to distinguish signals downstream of distinct chemoattractant GPCRs. Here we identify GflB, a Gα2 binding partner that directly couples the Dictyostelium cyclic AMP GPCR to Rap1. GflB localizes to the leading edge and functions as a Gα-stimulated, Rap1-specific guanine nucleotide exchange factor required to balance Ras and Rap signaling. The kinetics of GflB translocation are fine-tuned by GSK-3 phosphorylation. Cells lacking GflB display impaired Rap1/Ras signaling and actin and myosin dynamics, resulting in defective chemotaxis. Our observations demonstrate that GflB is an essential upstream regulator of chemoattractant-mediated cell polarity and cytoskeletal reorganization functioning to directly link Gα activation to monomeric G-protein signaling

Susceptibility to COPD:Differential Proteomic Profiling after Acute Smoking

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as "susceptible individuals". Here we perform unbiased analyses of proteomic profiles to assess how "susceptible individuals" differ from age-matched "non-susceptible individuals" in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD

The Response of Lactococcus lactis to Membrane Protein Production

Background: The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach. Methodology and Findings: Highly overproduced and poorly expressed membrane proteins both resulted in severe growth defects, whereas amplified levels of a soluble substrate receptor had no effect. In addition, membrane protein overproduction evoked a general stress response (upregulation of various chaperones and proteases), which is probably due to accumulation of misfolded protein. Notably, upon the expression of membrane proteins a cell envelope stress response, controlled by the two-component regulatory CesSR system, was observed. Conclusions: The physiological response of L. lactis to the overproduction of several membrane proteins was determined and compared to that of a soluble protein, thus offering better understanding of the bottlenecks related to membrane protein production and valuable knowledge for subsequent strain engineering.

Bacillus subtilis SpoIIIJ and YqjG Function in Membrane Protein Biogenesis

In all domains of life Oxa1p-like proteins are involved in membrane protein biogenesis. Bacillus subtilis, a model organism for gram-positive bacteria, contains two Oxa1p homologs: SpoIIIJ and YqjG. These molecules appear to be mutually exchangeable, although SpoIIIJ is specifically required for spore formation. SpoIIIJ and YqjG have been implicated in a posttranslocational stage of protein secretion. Here we show that the expression of either spoIIIJ or yqjG functionally compensates for the defects in membrane insertion due to YidC depletion in Escherichia coli. Both SpoIIIJ and YqjG complement the function of YidC in SecYEG-dependent and -independent membrane insertion of subunits of the cytochrome o oxidase and F1Fo ATP synthase complexes. Furthermore, SpoIIIJ and YqjG facilitate membrane insertion of F1Fo ATP synthase subunit c from both E. coli and B. subtilis into inner membrane vesicles of E. coli. When isolated from B. subtilis cells, SpoIIIJ and YqjG were found to be associated with the entire F1Fo ATP synthase complex, suggesting that they have a role late in the membrane assembly process. These data demonstrate that the Bacillus Oxa1p homologs have a role in membrane protein biogenesis rather than in protein secretion.