Tolerance and stress response to ethanol in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Eukaryotic cells have developed diverse strategies to combat the harmful effects of a variety of stress conditions. In the model yeast Saccharomyces cerevisiae, the increased concentration of ethanol, as the primary fermentation product, will influence the membrane fluidity and be toxic to membrane proteins, leading to cell growth inhibition and even death. Though little is known about the complex signal network responsible for alcohol stress responses in yeast cells, several mechanisms have been reported to be associated with this process, including changes in gene expression, in membrane composition, and increases in chaperone proteins that help stabilize other denatured proteins. Here, we review the recent progresses in our understanding of ethanol resistance and stress responses in yeast.     

Interaction with a lipid membrane: a key step in bacterial toxins virulence

Bacterial toxins are secreted as soluble proteins. However, they have to interact with a cell lipid membrane either to permeabilize the cells (pore forming toxins) or to enter into the cytosol to express their enzymatic activity (translocation toxins). The aim of this review is to suggest that the strategies developed by toxins to insert in a lipid membrane is mediated by their structure. Two categories, which contains both pore forming and translocation toxins, are emerging: alpha helical proteins containing hydrophobic domains and beta sheets proteins in which no hydrophobicity can be clearly detected. The first category would rather interact with the membrane through multi-spanning helical domains whereas the second category would form a beta barrel in the membrane.     

Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation

Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGD-restoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult.     

A signal sequence trap based on a constitutively active cytokine receptor.

Targeting of secreted and cell-surface proteins to the cell membrane is mediated by a short hydrophobic stretch of amino acids, termed the signal sequence. We have developed a method that detects signal sequences in cDNA fragments based on their ability to redirect a constitutively active mutant of a cytokine receptor to the cell surface, thereby permitting interleukin-3 (IL-3)-independent growth of Ba/F3 cells. Retrovirus-mediated expression of the fusions in IL-3-dependent cells was followed by selection of clones for growth in the absence of IL-3. Infection of cells with 5x10(6) viral particles in a pilot experiment led to the isolation of 150 known and 48 novel cDNA clones, and all the known cDNA clones were found to encode secreted and cell-surface proteins. In addition, we isolated type II membrane proteins, which have not been detected by existing signal sequence trap strategies.     

Emerging strategies in microbial haem capture

Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins. Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.     

Secretins of Pseudomonas aeruginosa: large holes in the outer membrane

Pseudomonas aeruginosa produces a large number of exoproteins, ranging from the ADP-ribosyltransferases exotoxin A and ExoS to degradative enzymes, such as elastase and chitinase. As it is a gram-negative bacterium, P. aeruginosa must be able to transport these exoproteins across both membranes of the cell envelope. In addition, also proteins that are part of cellular appendages, such as type IV pili and flagella, have to cross the cell envelope. Whereas the majority of the proteins transported across the inner membrane are dependent on the Sec channel, the systems for translocation across the outer membrane seem to be more diverse. Gram-negative bacteria have invented a number of different strategies during the course of evolution to achieve this goal. Although these transport machineries seem to be radically different, many of them actually depend on a member of the secretin protein family for their function. Recent results show that secretins form a large complex in the outer membrane, which constitutes the actual translocation channel. Understanding the working mechanism of this protein translocation channel could open up new strategies to target molecular machineries at the heart of many important virulence factors.     

Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers

Abstract

Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.     

Membrane Proteins: The Key Players of a Cancer Cell

Membrane proteins are involved in the prognosis of the most common forms of cancer. Membrane proteins are the hallmark of a cancer cell. The overexpressed membrane receptors are becoming increasingly important in cancer cell therapy. Current renewing therapy approaches based on receptor overexpression include; antibody therapy, nanocarrier drug delivery, and fluorescent tumor imaging in surgery. Gene profiling reveals cancer specific signatures and may identify membrane proteins that are related to cancer progression and lead to the development of improved therapy strategies in the future.     

A cell death assay for assessing the mitochondrial targeting of proteins

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.     

Endosome-to-Golgi transport pathways in physiological processes

The trans-Golgi network (TGN) is a major traffic hub of the cell, as it regulates membrane transport in the secretory pathway as well as receiving protein cargo by retrograde transport from endocytic compartments. Retrograde transport between endosomes and the TGN is essential for the recycling of membrane proteins which regulate a range of cellular and development functions. In addition, retrograde transport pathways are exploited by many bacterial toxins to mediate cytotoxicity and by some viral proteins to promote pathogenicity. Recent advances using a range of molecular cell biological strategies have identified multiple retrograde transport pathways each regulated by a distinct set of molecular machinery. Here we review recent advances in this field and highlight the importance of these transport pathways in a range of physiological processes.     

Overexpression of membrane proteins from higher eukaryotes in yeasts

Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug–target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.     

融合标签技术在膜蛋白结构研究中的应用

膜蛋白高级结构的研究包括不同的层次,即膜蛋白拓扑学结构的研究、利用核磁共振技术和蛋白质晶体衍射技术对三维结构的研究,以及膜蛋白复合体的研究。在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的蛋白质或多肽融合标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有融合标签的重组膜蛋自,不仅具有原有膜蛋白的功能活性,还具有融合标签所特有的生理生化特性,将会极大地促进膜蛋白结构和功能的研究。我们就目前膜蛋白结构研究中所涉及的融合标签技术及其应用策略和所取得的进展做一简述。     

Systematic analysis of native membrane protein complexes in Escherichia coli

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.     

Cell and process design for targeting of recombinant protein into the culture medium of <Emphasis Type="Italic">Escherichia coli</Emphasis>

This paper is a review of strategies to introduce protein into the liquid medium of Escherichia coli K-12 industrial production cells. The cell design strategies are generally based on one of two general mechanisms. The first strategy involves a two-stage translocation using active transporters in the cytoplasmic membrane followed by passive transport through the outer membrane. Passive transport is achieved through either external or internal destabilization of the E. coli structural components. The latter can be achieved by transplantation of destabilizing components (lysis proteins) that work by permeabilization of the outer membrane from the interior of the cell, or by using cells carrying mutations of structural components. Passive transport can also be achieved by a chemical, mechanical, or enzymatic permeabilization directed from outside the cell. The second strategy is realized through transplantation of proteins capable of active transport over one or both of the membranes. This involves the transplantation of secretion mechanisms into the K-12 cell from pathogenic E. coli as well as from other species. The process design strategies are dependent on environmental conditions and must take into account changes in physical parameters, medium design, and influx of limiting carbon source in fed-batch cultivation.     

Amphitropic proteins: regulation by reversible membrane interactions (review).

What do Src kinase, Ras-guanine nucleotide exchange factor, cytidylyltransferase, protein kinase C, phospholipase C, vinculin, and DnaA protein have in common? These proteins are amphitropic, that is, they bind weakly (reversibly) to membrane lipids, and this process regulates their function. Proteins functioning in transduction of signals generated in cell membranes are commonly regulated by amphitropism. In this review, the strategies utilized by amphitropic proteins to bind to membranes and to regulate their membrane affinity are described. The recently solved structures of binding pockets for specific lipids are described, as well as the amphipathic alpha-helix motif. Regulatory switches that control membrane affinity include modulation of the membrane lipid composition, and modification of the protein itself by ligand binding, phosphorylation, or acylation. How does membrane binding modulate the protein's function? Two mechanisms are discussed: (1) localization with the substrate, activator, or downstream target, and (2) activation of the protein by a conformational switch. This paper also addresses the issue of specificity in the cell membrane targetted for binding.     

Cell‐free expression profiling of E. coli inner membrane proteins

The high versatility and open nature of cell‐free expression systems offers unique options to modify expression environments. In particular for membrane proteins, the choice of co‐translational versus post‐translational solubilization approaches could significantly modulate expression efficiencies and even sample qualities. The production of a selection of 134 α‐helical integral membrane proteins of the Escherichia coli inner membrane proteome focussing on larger transporters has therefore been evaluated by a set of individual cell‐free expression reactions. The production profiles of the targets in different cell‐free expression modes were analyzed independently by three screening strategies. Translational green fluorescent protein fusions were analyzed as monitor for the formation of proteomicelles after cell‐free expression of membrane proteins in the presence of detergents. In addition, two different reaction configurations were implemented and performed either by robotic semi‐throughput approaches or by individually designed strategies. The expression profiles were specified for the particular cell‐free modes and overall, the production of 87% of the target list could be verified and approximately 50% could already be synthesized in preparative scales. The expression of several selected targets was up‐scaled to milliliter volumes and milligram amounts of production. As an example, the flavocytochrome YedZ was purified and its sample quality was demonstrated.     

Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.     

膜蛋白的拓扑学

膜蛋白的拓扑学是研究膜蛋白三维结构的出发点．利用融合蛋白和化学修饰等实验技术已确定了很多膜蛋白的拓扑学．对膜蛋白的转运与插膜的研究确定可能存在两类插膜元件．对已知拓扑学的膜蛋白的统计分析以及蛋白质工程的研究表明存在膜蛋白拓扑学的内正规则．目前已形成预测膜蛋白的拓扑学的比较可靠的策略,这在反向生物学上具有重要意义.但要进行三维结构的预测还有许多路要走．     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.     

Band 3 and ankyrin homologues are present in eye lens: evidence for all major erythrocyte membrane components in same non-erythroid cell

Although immunological homologues of erythrocyte membrane proteins have been individually discovered in a wide variety of tissues and cultured cells, the major structural components of the membrane have not yet been demonstrated simultaneously in the same cell type. Thus, considerable uncertainty continues to exist concerning whether the red cell homologues form elements of a structure which is similar to or unique from the framework which supports the erythrocyte membrane. Because the red cell cytoskeletal proteins, spectrin, actin and band 4.1, have been previously found in the superficial cortex of the lens, we decided to determine whether the corresponding membrane anchoring components of band 3 and ankyrin also occur in this cell type. Using antiserum specific for band 3 and ankyrin, we report the existence of immunologically cross-reactive proteins of similar molecular weight. Because these anchoring proteins appear and disappear coordinately with the aforementioned cytoskeletal proteins during the intermediate stages of lens cell maturation, it is conceivable that an erythrocyte-like membrane structural organization may occur transiently in the eye lens.