Corynebacterium glutamicum exhibits a membrane-related response to a small ferrocene-conjugated antimicrobial peptide

Multiresistant bacteria are becoming more and more widespread. It is therefore necessary to have new compound groups in hand, such as small cationic peptides, to cope with these challenges. In this work, we present a comprehensive approach by monitoring protein expression profiles in a Gram-positive bacterium (Corynebacterium glutamicum) to investigate the cellular response to such a compound, a ferrocene-conjugated arginine- and tryptophan-rich pentapeptide. To achieve this, a proteomic outline was performed where the compound-treated sample was compared with an untreated control. This study comprises more than 900 protein identifications, including numerous integral membrane proteins, and among these 185 differential expressions. Surprisingly, unregulated catalase and no elevated H₂O₂ levels demonstrate that no oxidative stress occurs after treatment with the iron-containing compound as a consequence of the potential Fenton reaction. A sufficient iron supply is evidenced by the iron-containing protein aconitase and SufB (the latter belongs to an iron–sulfur cluster assembly system) and decreased levels of ATP-binding-cassette-type cobalamin/Fe³⁺ siderophore transporters. The organometallic peptide antibiotic targets the cell membrane, which is evident by decreased levels of various integral membrane proteins, such as peptide permeases and transporters, and an altered lipid composition. Conversion to a more rigid cell membrane seems to be a relevant protective strategy of C. glutamicum against the ferrocene-conjugated antimicrobial peptide compound.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

Optimized proteomic analysis on gels of cell-cell adhering junctional membrane proteins

A high level of structural organization of functional membrane domains in very narrow regions of a plasma membrane is crucial for the functions of plasma membranes and various other cellular functions. Conventional proteomic analyses are based on total soluble cellular proteins. Thus, because of insolubility problems, they have major drawbacks for use in analyses of low-abundance proteins enriched in very limited and specific areas of cells, as well as in analyses of the membrane proteins in two-dimensional gels. We optimized proteomic analyses of cell-cell adhering junctional membrane proteins on gels. First, we increased the purity of cell-cell junctions, which are very limited and specific areas for cell-cell adhesion, from hepatic bile canaliculi. We then enriched junctional membrane proteins via a guanidine treatment; these became selectively detectable on two- dimensionally electrophoresed gels after treatment with an extremely high concentration of NP-40. The framework of major junctional integral membrane proteins was shown on gels. These included six novel junctional membrane proteins of type I, type II, and tetraspanin, which were identified by mass spectrometry and by a database sequence homology search, as well as 12 previously identified junctional membrane proteins, such as cadherins and claudins.     

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.     

In vivo analysis of integration of membrane proteins in Escherichia coli

The in vivo process of membrane protein integration was studied by pulse-labelling Escherichia coli cells, and assessing integral anchoring of labelled proteins to the lipid bilayer based on their resistance to alkali extraction. To conduct this experiment, conditions for extracting E. coli proteins with alkali were refined, and the immunoprecipitation procedures were improved to allow effective detection of integral membrane proteins. Examination of pulse-labelled, integral membrane proteins, including lactose permease (LacY), SecY, cytochrome omicron subunit II and leader peptidase revealed that all were in the alkali-insoluble fraction, indicating that membrane integration of these proteins takes place rapidly in wild-type cells. However, when LacY was synthesized in excess from a multicopy plasmid, significant proportions were found in the alkali-soluble fraction, indicating that the solubility in alkali is not an intrinsic property of the protein, and suggesting that LacY depends on some limited cellular factor for membrane integration. The unintegrated species of LacY sedimented slowly through an alkaline sucrose gradient. The secY24 mutant cells accumulated higher proportions of unintegrated LacY molecules at lower levels of overproduction than the sec+ cells. LacY overproduction in wild-type cells was found to inhibit processing (export) of beta-lactamase but not of OmpA and OmpF. These results are interpreted to mean that integration of LacY depends on multiple cellular components, one of which is also involved in export of beta-lactamase.     

Advances in qualitative and quantitative plant membrane proteomics

The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins. Along with their increased size, hydrophobicity, and the additional organelle and cellular features of plant cells relative to mammalian systems, the characterization of the plant membrane proteome presents unique challenges for effective qualitative and quantitative analysis using mass spectrometry (MS) analysis. Here, we present the latest advancements developed for the isolation and fractionation of plant organelles and their membrane components amenable to MS analysis. Separations of membrane proteins from these enriched preparations that have proven effective are discussed for both gel- and liquid chromatography-based MS analysis. In this context, quantitative membrane proteomic analyses using both isotope-coded and label-free approaches are presented and reveal the potential to establish a wider-biological interpretation of the function of plant membrane proteins that will ultimately lead to a more comprehensive understanding of plant physiology and their response mechanisms.     

Sub-proteomic study on macrophage response to Candida albicans unravels new proteins involved in the host defense against the fungus

In previous proteomic studies on the response of murine macrophages against Candida albicans, many differentially expressed proteins involved in processes like inflammation, cytoskeletal rearrangement, stress response and metabolism were identified. In order to look for proteins important for the macrophage response, but in a lower concentration in the cell, 3 sub-cellular extracts were analyzed: cytosol, organelle/membrane and nucleus enriched fractions from RAW 264.7 macrophages exposed or not to C. albicans SC5314 for 3 h. The samples were studied using DIGE technology, and 17 new differentially expressed proteins were identified. This sub-cellular fractionation permitted the identification of 2 mitochondrion proteins, a membrane receptor, Galectin-3, and some ER related proteins, that are not easily detected in total cell extracts. Besides, the study of different fractions allowed us to detect, not only total increase in Galectin-3 protein amount, but its distinct allocation along the interaction. The identified proteins are involved in the pro-inflammatory and oxidative responses, immune response, unfolded protein response and apoptosis. Some of these processes increase the host response and others could be the effect of C. albicans resistance to phagocytosis. Thus, the sub-proteomic approach has been a very useful tool to identify new proteins involved in macrophage-fungus interaction. This article is part of a Special Issue entitled: Translational Proteomics.     

Key phases in the formation of caveolae

Caveolae are abundant plasma membrane pits formed by the coordinated action of peripheral and integral membrane proteins and membrane lipids. Here, we discuss recent studies that are starting to provide a glimpse of how filamentous cavin proteins, membrane-embedded caveolin proteins, and specific plasma membrane lipids are brought together to make the unique caveola surface domain. Protein assembly involves multiple low-affinity interactions that are dependent on ‘fuzzy’ charge-dependent interactions mediated in part by disordered cavin and caveolin domains. We propose that cavins help generate a lipid domain conducive to full insertion of caveolin into the bilayer to promote caveola formation. The synergistic assembly of these dynamic protein complexes supports the formation of a metastable membrane domain that can be readily disassembled both in response to cellular stress and during endocytic trafficking. We present a mechanistic model for generation of caveolae based on these new insights.     

Phosphatidylinositol transfer proteins: negotiating the regulatory interface between lipid metabolism and lipid signaling in diverse cellular processes

Phosphoinositides represent only a small percentage of the total cellular lipid pool. Yet, these molecules play crucial roles in diverse intracellular processes such as signal transduction at membrane-cytosol interface, regulation of membrane trafficking, cytoskeleton organization, nuclear events, and the permeability and transport functions of the membrane. A central principle in such lipid-mediated signaling is the appropriate coordination of these events. Such an intricate coordination demands fine spatial and temporal control of lipid metabolism and organization, and consistent mechanisms for specifically coupling these parameters to dedicated physiological processes. In that regard, recent studies have identified Sec14-like phosphatidylcholine transfer protein (PITPs) as "coincidence detectors," which spatially and temporally link the diverse aspects of the cellular lipid metabolome with phosphoinositide signaling. The integral role of PITPs in eukaryotic signal transduction design is amply demonstrated by the mammalian diseases associated with the derangements in the function of these proteins, to stress response and developmental regulation in plants, to fungal dimorphism and pathogenicity, to membrane trafficking in yeast, and higher eukaryotes. This review updates the recent advances made in the understanding of how these proteins, specifically PITPs of the Sec14-protein superfamily, operate at the molecular level and further describes how this knowledge has advanced our perception on the diverse biological functions of PITPs.     

Biotechnical applications of small heat shock proteins from bacteria

The stress responses of most bacteria are thought to involve the upregulation of small heat shock proteins. We describe here some of the most pertinent aspects of small heat shock proteins, to highlight their potential for use in various applications. Bacterial species have between one and 13 genes encoding small heat shock proteins, the precise number depending on the species considered. Major efforts have recently been made to characterize the protein protection and membrane stabilization mechanisms involving small heat shock proteins in bacteria. These proteins seem to be involved in the acquisition of cellular heat tolerance. They could therefore potentially be used to maintain cell viability under unfavorable conditions, such as heat shock or chemical treatments. This review highlights the potential roles of applications of small heat shock proteins in stabilizing overproduced heterologous proteins in Escherichia coli, purified bacterial small heat shock proteins in protein biochip technology, proteomic analysis and food technology and the potential impact of these proteins on some diseases. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Phosphorylation dynamics of membrane proteins from Arabidopsis roots submitted to salt stress

An excess of NaCl in the soil is detrimental for plant growth. It interferes with mineral nutrition and water uptake and leads to accumulation of toxic ions in the plant. Understanding the response of roots to NaCl stress may facilitate the development of crops with increased tolerance to this and other stresses. Since controls achieved at the posttranslational level are of critical importance for regulating protein function, the present work used a robust label‐free quantitative proteomic methodology to quantify phosphorylation events that affect root membrane proteins in Arabidopsis, in response to short‐term (up to 2 h) NaCl treatments. This work identified 302 proteotypic phosphopeptides including 77 novel phosphorylated sites. NaCl treatment significantly altered the abundance of 74 phosphopeptides, giving novel insights into the regulation of major classes of membrane proteins, including ATPases, sodium transporters, and aquaporins. The data provide a unique access to phosphorylation reprogramming of ionic equilibrium in plant cells under NaCl stress. The use of predictive bioinformatic tools for kinase motifs suggested that root membrane proteins are substrates of cAMP‐dependent protein kinase, cGMP‐dependent protein kinase, and protein kinase C families, also called AGC kinases, arguing for an important role of lipid signaling in abiotic stress responses. It also pointed to cross‐talks between protein kinase families during NaCl stress.     

Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytochrome bc(1) complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSP1467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment-protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.     

Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation

Lipid droplet is a cellular organelle with a neutral lipid core surrounded by a phospholipid monolayer and coated with structural as well as functional proteins. The determination of these proteins, especially their functional regulations and dynamic movement on and off droplets, holds a key to resolving the biological functions of the cellular organelle. To address this, we carried out a comprehensive proteomic study that includes a complete proteomic, a phosphoprotein proteomic, and a comparative proteomic analysis using purified lipid droplets and mass spectrometry techniques. The complete proteome identified 125 proteins of which 70 proteins had not been identified on droplets of mammalian cells previously. In phosphoprotein proteomic analysis, 7 functional lipid droplet proteins were determined to be phosphorylated, including adipose differentiation related protein (ADRP/ADFP), two Rab proteins, and four lipid metabolism enzymes, including adipose triglyceride lipase (ATGL). To understand the dynamics of lipid droplets, GTP-dependent protein recruitment was analyzed by comparative proteomics. Arf1 and some of its coatomers, three other Arfs, several other small G-proteins including 3 Rabs, and several lipid synthetic enzymes were recruited from cytosol to purified droplets. Together, the present study suggests that lipid droplet is an active and dynamic cellular organelle that governs lipid homeostasis and intracellular trafficking through protein phosphorylation as well as GTP-regulated protein translocation.     

HSPB1 influences mitochondrial respiration in ER-stressed beta cells

Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.     

Transient repression of the synthesis of OmpF and aspartate transcarbamoylase in Escherichia coli K12 as a response to pollutant stress

Abstract The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride. Changes in protein patterns were identified after pulse-chase labelling of proteins with [³⁵S]methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE). We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred. Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase). Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress.