Transmembrane-Domain Trapping: A Novel Method for Isolation of cDNAs Encoding Putative Membrane Proteins

We have developed a method that enables us to isolate cDNAsof putative membrane proteins. The system is designed to isolatea cDNA which can provide the transmembrane domain to the extracellularpart of the IL-2 receptor chain. We constructed a p18Mac vectorby putting part of the IL-2 receptor chain cDNA that encodedits signal sequence and extracellular domain, a cDNA cloningsite and a poly(A) additional signal after a strong promoterSR. If a cloned cDNA provides a transmembrane domain in-frame,the extracellular domain of the IL-2 receptor chain will beexpressed on the surface of the transfected cells. Otherwise,the chimeric protein will be either secreted or retained insidethe transfected cells. We made a cDNA library using p18Mac andscreened for cDNA clones which allowed the expression of theextracellular domain of the IL-2 receptor chain on the cellsurface. Of the 2000 clones screened, 5 clones were scored aspositive. Partial sequence analysis revealed that one cloneencoded the amyloid precursor protein, two others encoded mitochondrialproteins and the rest were new. These results suggest the systemis effective in isolating cDNAs encoding putative membrane proteins.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Cytoskeletal disruption and small heat shock protein translocation immediately after lengthening contractions

The purposes of this study were to determine whether, immediately after lengthening contractions, 1) levels of specific force-transmitting cytoskeletal elements are reduced in skeletal muscle cells and 2) cytosolic small heat shock proteins (HSPs) translocate to structures prone to disruption. Western blot analysis demonstrated decreased concentrations of z-disk proteins -actinin and plectin and membrane scaffolding proteins dystrophin and -spectrin in muscle exposed to lengthening contractions compared with contralateral control muscle. Lengthening contractions also resulted in immediate translocation of constitutively expressed HSP25 and B-crystallin from the soluble to the insoluble fraction of muscle homogenates, and cryosections showed translocation from a diffuse, cytosolic localization to striations that corresponded to z-disks. Lengthening contraction-induced translocation of HSP25 and B-crystallin was associated with phosphorylation of these small HSPs, which may trigger their protective activity. In summary, these findings demonstrate loss of z-disk and membrane scaffolding proteins immediately after lengthening contractions, and concomitant translocation of HSP25 and B-crystallin to the z-disk, which may help to stabilize or repair cytoskeletal elements at this site. skeletal muscle injury; heat shock protein 25; B-crystallin; dystrophin; desmin     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins

The recombinant expression of integral membrane proteins is considered a major challenge, and together with the crystallization step, the major hurdle toward routine structure determination of membrane proteins. Basic methodologies for high-throughput (HTP) expression optimization of soluble proteins have recently emerged, providing statistically significant success rates for producing such proteins. Experimental procedures for handling integral membrane proteins are generally more challenging, and there have been no previous comprehensive reports of HTP technology for membrane protein production. Here, we present a generic and integrated parallel HTP strategy for cloning and expression screening of membrane proteins in their detergent solubilized form. Based on this strategy, we provide overall success rates for membrane protein production in Escherichia coli, as well as initial benchmarking statistics of parameters such as expression vectors, strains, and solubilizing detergents. The technologies were applied to 49 E. coli integral membrane proteins with human homologs and revealed that 71% of these proteins could be produced at sufficient levels to allow milligram amounts of protein to be relatively easily purified, which is a significantly higher success rate than anticipated. We attribute the high success rate to the quality and robustness of the methodology used, and to introducing multiple parameters such as different vectors, strains, and detergents. The presented strategy demonstrates the usefulness of HTP technologies for membrane protein production, and the feasibility of large-scale programs for elucidation of structure and function of bacterial integral membrane proteins.     

The influence of Lyn kinase on Na,K-ATPase in porcine lens epithelium

Na,K-ATPase is essential for the regulation of cytoplasmic Na⁺ and K⁺ levels in lens cells. Studies on the intact lens suggest activation of tyrosine kinases may inhibit Na,K-ATPase function. Here, we tested the influence of Lyn kinase, a Src-family member, on tyrosine phosphorylation and Na,K-ATPase activity in membrane material isolated from porcine lens epithelium. Western blot studies indicated the expression of Lyn in lens cells. When membrane material was incubated in ATP-containing solution containing partially purified Lyn kinase, Na,K-ATPase activity was reduced by 38%. Lyn caused tyrosine phosphorylation of multiple protein bands. Immunoprecipitation and Western blot analysis showed Lyn treatment causes an increase in density of a 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase ₁ polypeptide. Incubation with protein tyrosine phosphatase 1B (PTP-1B) reversed the Lyn-dependent tyrosine phosphorylation increase and the change of Na,K-ATPase activity. The results suggest that Lyn kinase treatment of a lens epithelium membrane preparation is able to bring about partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase ₁ catalytic subunit. lens; Na,K-ATPase; tyrosine phosphorylation; Lyn     

Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells

Cells expressingconnexin43 are able to upregulate gap junction (GJ) communication byenhancing the assembly of new GJs, apparently through increasedconnexin trafficking. Because G proteins are known to regulatedifferent aspects of protein trafficking, we examined the effects ofpertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJassembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicatedby a reduction in dye transfer. Electron microscopy also revealed a60% decrease in the number of GJ channels per cell interface.Importantly, PTX blocked the twofold enhancement in GJ assembly foundin the presence of low-density lipoprotein. Two G_iproteins (G_i2 and G_i3), which have beenimplicated in the control of membrane trafficking, reacted with PTX inADP-ribosylation studies. PTX and/or the trafficking inhibitors,brefeldin A and monensin, inhibited GJ assembly to comparable degrees.In addition, assays for GJ hemichannels demonstrated reduced plasmamembrane levels of connexin43 following PTX treatment. These resultssuggest that PTX-sensitive G proteins regulate connexin43 trafficking,and, as a result of inhibition with PTX, the number of plasma membranehemichannels available for GJ assembly is reduced.

     

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.     

Spatiotemporal expression of catenins, ZO-1, and occludin during early polarization of hepatic WIF-B9 cells

WIF-B9 is a suitablemodel for in vitro studies of hepatocyte polarity. To better understandpolarity establishment, we have localized key proteins of the adhesionsystem, cytoskeleton, and tight junctions soon after plating, when mostcells are isolated or in doublets. In isolated attached cells, onlycytoskeletal proteins (tubulin, cytokeratins) displayed a preciselocalization. As soon as two cells formed a doublet, E-cadherin, -,-, and -catenins, and p120 protein were present at the doubletcontiguous membrane. Actin, ezrin, and zonula occludens-1 (ZO-1)colocalized at this membrane, but not in all doublets: ezrin waspresent only at contiguous membrane expressing ZO-1, and ZO-1 waspresent only at membrane expressing actin. In contrast, occludin wasspread throughout the doublet cytoplasm. With time in culture, these proteins localized transiently, as in cells expressing simple epithelial polarity, and finally, as in hepatocytes. We conclude thatduring WIF-B9 early polarization, key proteins are settled according toa hierarchy, as has been shown for Madin-Darby canine kidney cells.Cytoplasmic complexes of E-cadherin-catenin were detected during thewhole polarization process; they were more abundant in fully polarized cells.

     

Cholinergic receptor-mediated differential cytoskeletal recruitment of actin- and integrin-binding proteins in intact airway smooth muscle

We tested the hypothesis that cholinergic receptor stimulation recruits actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex in intact airway smooth muscle. We stimulated bovine tracheal smooth muscle with carbachol and fractionated the tissue homogenate into pellet (P) and supernatant (S) by ultracentrifugation. In unstimulated tissues, calponin exhibited the highest basal P-to-S ratio (P/S; 2.74 ± 0.47), whereas vinculin exhibited the lowest P/S (0.52 ± 0.09). Cholinergic receptor stimulation increased P/S of the following proteins in descending order of sensitivity: -actinin > talin metavinculin > -smooth muscle actin > vinculin calponin. Carbachol induced ERK1/2 phosphorylation by 300% of basal value. U0126 (10 µM) completely inhibited carbachol-induced ERK1/2 phosphorylation but did not significantly affect the correlation between -actinin P/S and carbachol concentration. This observation indicates that cytoskeletal/membrane recruitment of -actinin is independent of ERK1/2 mitogen-activated protein kinase activation. Metavinculin and vinculin are splice variants of a single gene, but metavinculin P/S was significantly higher than vinculin P/S. Furthermore, the P/S of metavinculin but not vinculin increased significantly in response to cholinergic receptor stimulation. Calponin and -actinin both belong to the family of calponin homology (CH) domain proteins. However, unlike -actinin, the calponin P/S did not change significantly in response to cholinergic receptor stimulation. These findings indicate differential cytoskeletal/membrane recruitment of actin- and integrin-binding proteins in response to cholinergic receptor stimulation in intact airway smooth muscle. -Actinin, talin, and metavinculin appear to be key cytoskeletal proteins involved in the recruitment process. actinin; mitogen-activated protein kinase; metavinculin; vinculin     

HMPAS: Human Membrane Protein Analysis System

Background

Membrane proteins perform essential roles in diverse cellular functions and are regarded as major pharmaceutical targets. The significance of membrane proteins has led to the developing dozens of resources related with membrane proteins. However, most of these resources are built for specific well-known membrane protein groups, making it difficult to find common and specific features of various membrane protein groups.

Methods

We collected human membrane proteins from the dispersed resources and predicted novel membrane protein candidates by using ortholog information and our membrane protein classifiers. The membrane proteins were classified according to the type of interaction with the membrane, subcellular localization, and molecular function. We also made new feature dataset to characterize the membrane proteins in various aspects including membrane protein topology, domain, biological process, disease, and drug. Moreover, protein structure and ICD-10-CM based integrated disease and drug information was newly included. To analyze the comprehensive information of membrane proteins, we implemented analysis tools to identify novel sequence and functional features of the classified membrane protein groups and to extract features from protein sequences.

Results

We constructed HMPAS with 28,509 collected known membrane proteins and 8,076 newly predicted candidates. This system provides integrated information of human membrane proteins individually and in groups organized by 45 subcellular locations and 1,401 molecular functions. As a case study, we identified associations between the membrane proteins and diseases and present that membrane proteins are promising targets for diseases related with nervous system and circulatory system. A web-based interface of this system was constructed to facilitate researchers not only to retrieve organized information of individual proteins but also to use the tools to analyze the membrane proteins.

Conclusions

HMPAS provides comprehensive information about human membrane proteins including specific features of certain membrane protein groups. In this system, user can acquire the information of individual proteins and specified groups focused on their conserved sequence features, involved cellular processes, and diseases. HMPAS may contribute as a valuable resource for the inference of novel cellular mechanisms and pharmaceutical targets associated with the human membrane proteins. HMPAS is freely available at http://fcode.kaist.ac.kr/hmpas.

     

Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis

Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the σ^W regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.Membrane-embedded proteins are crucial for cellular homeostasis and life. Membrane proteins generally account for about 30% of the open reading frames in both prokaryotic and eukaryotic genomes (49), and they are involved in a wide range of different tasks. These include vital processes, such as energy transduction, phospholipid biosynthesis, protein translocation, cell wall biogenesis, cell division, and control of cell shape (52). Importantly, membrane proteins are partially exposed to the extracytoplasmic environment, which makes them readily accessible to drugs. For this reason, membrane proteins have become a major class of proteins in terms of current drug targets. Essential membrane proteins, which are indispensable for cell proliferation under specific conditions, are especially interesting from the pharmaceutical and biomedical perspectives because they represent prime targets for chemotherapy.Unfortunately, progress in the area of membrane protein research has so far been slow. This has been attributed primarily to the high hydrophobicity of membrane proteins, which complicates high-level production, purification, and crystallization (25). Consequently, yields are often frustratingly low, as underscored by a series of elegant screens for membrane protein overproduction in Escherichia coli (10, 11, 15, 47). Moreover, the accumulation of overproduced proteins in biological membranes may affect bilayer integrity, which would be toxic for the producing cell (33). Additional limitations are potentially caused by saturation of the cellular machinery for insertion of proteins into the membrane or by saturation of the membrane itself, resulting in the cytoplasmic accumulation of overproduced membrane proteins as well as native membrane proteins (46). Such overproduced proteins are usually misfolded and/or inactive, and they have a high tendency to form insoluble (micro)aggregates. These practical problems focus attention on the fundamental question of which cellular mechanisms set the key limits to membrane protein production.In the present studies, we show that important problems in membrane protein overproduction can be overcome by using different strains of the gram-positive bacterium Bacillus subtilis as the expression host, and we identify two key mechanisms that set limits to membrane protein production in this organism. B. subtilis is highly appreciated for biotechnological applications because it has a large capacity to secrete high-quality proteins into the culture medium and because it has the status of generally recognized as safe (18, 38, 50). Furthermore, B. subtilis is amenable to genetic engineering, and many expression systems are available (2, 16, 31, 40, 43, 44). This prompted us to investigate whether the secretion machinery of B. subtilis, which is also involved in membrane protein biogenesis (52), can be exploited for membrane protein overproduction. As model proteins for our studies, we selected essential membrane proteins that have a good potential to serve as targets for novel antimicrobial drugs. Accordingly, we not only overproduced B. subtilis membrane proteins but also their orthologues from the important human pathogen Staphylococcus aureus. Studies on these essential proteins are considered to be of major relevance, since S. aureus is rapidly gaining resistance against all available antibiotics and novel antibiotics against this pathogen are urgently needed (7, 17). The results of the present studies with homologous membrane proteins from B. subtilis and S. aureus show that, like in other expression hosts, bottlenecks in membrane protein production also do exist in B. subtilis. Importantly, however, at least some of the encountered bottlenecks can be overcome, because they relate to two dispensable membrane-associated stress-responsive systems: the σ^W regulon and the CssRS two-component regulatory system. Thus, the removal of at least one of these stress-responsive systems can result in drastically improved yields of particular membrane proteins.     

An approach to systematic detection of protein structural motifs

A procedure to detect similar local structures of proteins fromC coordinates is presented. First, the conformations of seven-residuepeptide segments are approximated by a limited number of representatives,each of which is assigned a symbol. Thus, the overall conformationof a protein is represented by a symbol string. The comparisonof these symbol strings using a sequence alignment techniquethen gives pairs of similar local structures. These pairs areconsidered candidates of structural motifs. The applicationof the procedure to the analysis of 93 proteins gave 858 pairsof similar local structures, which included several well-knownstructural motifs such as the nucleotide-binding ßß-unitand the calcium-binding EF hand. The characterization of aminoacid patterns of similar local structures given by the procedureshould be useful for the development of protein structure predictionbased on the acquisition of empirical rules from a large-scaledatabase.     

The early stages of the intracellular transport of membrane proteins: clinical and pharmacological implications

Intracellular transport mechanisms ensure that integral membrane proteins are delivered to their correct subcellular compartments. Efficient intracellular transport is a prerequisite for the establishment of both cell architecture and function. In the past decade, transport processes of proteins have also drawn the attention of clinicians and pharmacologists since many diseases have been shown to be caused by transport-deficient proteins. Membrane proteins residing within the plasma membrane are transported via the secretory (exocytotic) pathway. The general transport routes of the secretory pathway are well established. The transport of membrane proteins starts with their integration into the ER membrane. The ribosomes synthesizing membrane proteins are targeted to the ER membrane, and the nascent chains are co-translationally integrated into the bilayer, i.e., they are inserted while their synthesis is in progress. During ER insertion, the orientation (topology) of the proteins in the membrane is determined. Proteins are folded, and their folding state is checked by a quality control system that allows only correctly folded forms to leave the ER. Misfolded or incompletely folded forms are retained, transported back to the cytosol and finally subjected to proteolysis. Correctly folded proteins are transported in the membranes of vesicles through the ER/Golgi intermediate compartment (ERGIC) and the individual compartments of the Golgi apparatus (cis, medial, trans) to the plasma membrane. In this review, the current knowledge of the first stages of the intracellular trafficking of membrane proteins will be summarized. This early secretory pathway includes the processes of ER insertion, topology determination, folding, quality control and the transport to the Golgi apparatus. Mutations in the genes of membrane proteins frequently lead to misfolded forms that are recognized and retained by the quality control system. Such mutations may cause inherited diseases like cystic fibrosis or retinitis pigmentosa. In the second part of this review, the clinical implications of the early secretory pathway will be discussed. Finally, new pharmacological strategies to rescue misfolded and transport-defective membrane proteins will be outlined.Abbreviations AP1 Clathrin-associated adaptor protein complex 1 - AQP Aquaporin - ARF ADP-ribosylation factor - AVP 8-Arginine-vasopressin;BiP immunoglobulin heavy chain binding protein - CFTR Cystic fibrosis transmembrane conductance regulator - CLQTS Congenital long QT syndrome - CMT Charcot-Marie-Tooth syndrome - CNX Calnexin - COPI Coat protein complex I - COPII Coat protein complex II - CPX 8-Cyclopentyl-1,2-dipropylxanthine - CRT Calreticulin - CSID Congenital sucrose-isomaltase deficiency - Cx Connexin - cGMP Cyclic 3:5 guanosine monophosphate - ECL Extracellular loop - EndoH Endoglycosidase H - ER Endoplasmic reticulum - ERAD ER-associated degradation - ERGIC ER/Golgi intermediate compartment - ERp ER protein - ET_BR Human endothelin B receptor - FH Familial hypercholesterolemia - GABA Gamma amino butyric acid - GFP Green fluorescent protein - GH Growth hormone - GHIS Growth hormone insensitivity syndrome - GLCase Glucosidase - GlcNac N-acetylglucosamine - GPCR G protein-coupled receptor - GPI Glycosylphosphatidylinositol - G protein GTP-binding protein - GRP Glucose-regulated protein - HA Hemagglutinin - Hdj-2 Human DnaJ-2 protein - HFE Human hemochromatosis protein - HH Hereditary hemochromatosis - HEK 293 cells Human embryonic kidney 293 cells - HERG Human ether-a-go-go-related protein - Hsc70 Heat shock cognate 70 protein - ICL Intracellular loop - IGF-I Insulin-like growth factor-1 - I_Kr Rapidly activating delayed rectifier potassium current - I_Ks Slowly activating delayed rectifier potassium current - JAK Janus kinase - LDL Low-density lipoprotein - LH Luteinizing hormone/choriogonadotropin - LS Laron syndrome - MATP Membrane associated transporter protein - MDCK cells Madin-Darby canine kidney epithelial cells - MHC Major histocompatibility complex - MiRP1 minK-related peptide 1 - NDI Congenital nephrogenic diabetes insipidus - NMDA N-methyl-d-aspartate - OCA Oculocutaneous albinism - PDI Protein disulfide isomerase - Pgp P-glycoprotein - PKA Protein kinase A - PLP Proteolipid protein - PMP22 Peripheral myelin protein 22 - RP Primary retinitis pigmentosa - SI Sucrase-isomaltase - SNARE Ethylmaleimide-sensitive factor attachment protein - SRP Signal recognition particle - TCR T-cell antigen receptor - TM Transmembrane domain - TRAM Translocating chain-associated membrane protein - Tyr Tyrosinase - Tyrp1 Tyrosinase-related protein-1 - UGGT UDP-glucose:glycoprotein glucosyltransferase - VIP Vesicular-integral membrane protein - V₂R Vasopressin V₂ receptor - VSV Vesicular stomatitis virus     

Application of a High Throughput Method of Biomarker Discovery to Improvement of the EarlyCDT?-Lung Test

Background

The National Lung Screening Trial showed that CT screening for lung cancer led to a 20% reduction in mortality. However, CT screening has a number of disadvantages including low specificity. A validated autoantibody assay is available commercially (EarlyCDT®-Lung) to aid in the early detection of lung cancer and risk stratification in patients with pulmonary nodules detected by CT.Recent advances in high throughput (HTP) cloning and expression methods have been developed into a discovery pipeline to identify biomarkers that detect autoantibodies. The aim of this study was to demonstrate the successful clinical application of this strategy to add to the EarlyCDT-Lung panel in order to improve its sensitivity and specificity (and hence positive predictive value, (PPV)).

Methods and Findings

Serum from two matched independent cohorts of lung cancer patients were used (n = 100 and n = 165). Sixty nine proteins were initially screened on an abridged HTP version of the autoantibody ELISA using protein prepared on small scale by a HTP expression and purification screen. Promising leads were produced in shake flask culture and tested on the full assay. These results were analyzed in combination with those from the EarlyCDT-Lung panel in order to provide a set of re-optimized cut-offs. Five proteins that still displayed cancer/normal differentiation were tested for reproducibility and validation on a second batch of protein and a separate patient cohort. Addition of these proteins resulted in an improvement in the sensitivity and specificity of the test from 38% and 86% to 49% and 93% respectively (PPV improvement from 1 in 16 to 1 in 7).

Conclusion

This is a practical example of the value of investing resources to develop a HTP technology. Such technology may lead to improvement in the clinical utility of the EarlyCDT­-Lung test, and so further aid the early detection of lung cancer.     

Probing FhuA''-''PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps. Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling. Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression. The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins. In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured. For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands. However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate. In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate. This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.Download video file.^{(127M, mp4)}     

Mechanisms of Protein Sorting in Mitochondria

A protein’s function is intimately linked to its correct subcellular location, yet the machinery required for protein synthesis is predominately cytosolic. How proteins are trafficked through the confines of the cell and integrated into the appropriate cellular compartments has puzzled and intrigued researchers for decades. Indeed, studies exploring this premise revealed elaborate cellular protein translocation and sorting systems, which ensure that all proteins are shuttled to the appropriate cellular destination, where they fulfill their specific functions. This holds true for mitochondria, where sophisticated molecular machines serve to recognize incoming precursor proteins and integrate them into the functional framework of the organelle. We summarize the recent progress in our understanding of mitochondrial protein sorting and the machineries and mechanisms that mediate and regulate this highly dynamic cellular process essential for survival of virtually all eukaryotic cells.Mitochondria are multifunctional double-membrane-bound organelles that arose from a bacterial endosymbiont during the evolution of eukaryotic cells. Known as the powerhouses of the cell, mitochondria harbor the oxidative phosphorylation machinery for ATP synthesis, but also a large number of biosynthetic pathways. Moreover, they are intimately involved in complex cellular processes, like calcium homeostasis and programmed cell death. As a relic of their evolutionary origin, mitochondria contain their own genetic material and machineries to manufacture their own RNAs and proteins. However, the small circular mitochondrial genome encodes only a few proteins (8 and 13 polypeptides in yeast and humans, respectively). All remaining mitochondrial proteins (approximately 99%) are encoded by the nuclear genome and synthesized on cytosolic ribosomes in their precursor forms. To acquire their mature, functional state these precursor proteins need to be efficiently targeted and imported into mitochondria and sorted to the correct submitochondrial compartment: outer membrane, intermembrane space (IMS), inner membrane, and matrix. The inner mitochondrial membrane is further subdivided into the inner boundary membrane, which is closely opposed to the outer membrane, and large tubular invaginations, termed cristae membranes. Within the four mitochondrial compartments, sophisticated translocation, sorting, and assembly machineries serve to establish incoming precursors in a functional state within the context of their new environment. Advances in the last decade, particularly because of the application of proteomic approaches, have significantly extended the number of components and machineries known to be involved in mitochondrial protein import (Sickmann et al. 2003; Prokisch et al. 2004; Reinders et al. 2006; Pagliarini et al. 2008). These and previous discoveries have provided us with the current framework, which suggests the presence of at least six distinct translocation and assembly machineries within mitochondria (Fig. 1). In this article, we will summarize our current understanding of the machineries for mitochondrial protein import and describe the different molecular mechanisms that execute this essential task.Open in a separate windowFigure 1.Overview of mitochondrial protein sorting pathways. Cytosolic chaperones deliver precursor proteins to the organelle in a translocation-competent state. Some α-helical proteins are inserted into the outer membrane with the help of Mim1. Virtually all other precursors initially traverse the outer membrane via the TOM complex and are subsequently routed to downstream sorting pathways. Biogenesis of outer membrane β-barrel proteins requires the small TIM chaperones of the IMS and the SAM complex. Cysteine-containing IMS proteins are imported via the MIA pathway. Metabolite carriers of the inner mitochondrial membrane are transferred by the small TIM chaperones to the TIM22 complex, which mediates their membrane integration. Presequence-containing precursors are directly taken over from the TOM complex by the TIM23 machinery that either inserts these proteins into the membrane or translocates them into the matrix in cooperation with the import motor PAM. OM, outer membrane; IMS, intermembrane space; IM, inner membrane, Δψ, membrane potential across the inner mitochondrial membrane.