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PubMed2004

Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity.

We report the development and characterization of a polyacrylamide-based protein immobilization strategy for surface-bound protein assays, including concentration detection, binding affinity, and enzyme kinetics. Glutathione S-transferase (GST) fusion proteins have been labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through copolymerization with acrylic monomer. The specific attachment of GST-green fluorescent protein (GFP) fusion protein was more than sevenfold greater than the nonspecific attachment of nonacrylic-labeled GST-GFP; 0.32 ng/mm(2) of surface-attached GST-GFP was detectable by direct measurement of GFP fluorescence and this lower detection limit was reduced to 0.080 ng/mm(2) using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src. Michaelis-Menten kinetic constants for the reaction occurring in solution were K(m) = 2.7 +/- 1.0 microM and V(max) = 8.1 +/- 3.1 (arbitrary units). Kinetic values for the reaction utilizing surface-immobilized substrate were K(m) = 0.36 +/- 0.033 microM and V(max) = 9.7 +/- 0.63 and were found to be independent of the acrylamide concentration within the copolymer. Such a surface attachment strategy should be applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.

Acrylic Resins↗
PubMed2006

Landscape of the hnRNP K protein-protein interactome.

The heterogeneous nuclear ribonucleoprotein K is an ancient RNA/DNA-binding protein that is involved in multiple processes that compose gene expression. The pleiotropic action of K protein reflects its ability to interact with different classes of factors, interactions that are regulated by extracellular signals. We used affinity purification and MS to better define the repertoire of K protein partners. We identified a large number of new K protein partners, some typically found in subcellular compartments, such as plasma membrane, where K protein has not previously been seen. Electron microscopy showed K protein in the nucleus, cytoplasm, mitochondria, and in vicinity of plasma membrane. These observations greatly expanded the view of the landscape of K protein-protein interaction and provide new opportunities to explore signal transduction and gene expression in several subcellular compartments.

Animals↗
PubMed2006

An automatable screen for the rapid identification of proteins amenable to refolding.

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

Animals↗
PubMed2000

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters.

The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.

Base Sequence↗
PubMed2004

Genome-scale, biochemical annotation method based on the wheat germ cell-free protein synthesis system.

Since the complete genomic DNA sequencing of various species, attention has turned to the structural properties, and functional characteristics of proteins. Current cell-free protein expression systems from eukaryotes are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. This report reviews the high-throughput, genome-scale biochemical annotation method based on the cell-free system prepared from wheat embryos. We first briefly reviewed our highly efficient and robust wheat germ cell-free protein synthesis system, and then showed an application of the system for materialization and characterization of genetic information taking a cDNA library of protein kinase from Arabidopsis thaliana as an example. The procedure consists of: (1) fusion of the gene-of-interest to a purification-tag, amplified by the split-primer PCR method; (2) transcription and purification of mRNA; (3) cell-free protein synthesis in the bilayer system using 96-well titer plate; (4) affinity purification and activity measurement. We took 439 cDNAs encoding kinases among 1064 genes annotated so far, and they were translated in parallel into protein. Subsequent assay revealed 207 products having autophosphorylation activity. Furthermore, seven proteins out of 26 calcium-dependent protein kinase genes tested did phosphorylate a synthetic peptide substrate in the presence of calcium ion, demonstrating that the translation products, retained their substrate specificity. The information on biochemical function of gene products accumulated should revolutionize our understanding of biology and fundamentally alter the practice of medicine and influence other industries as well.

Arabidopsis↗
PubMedDate not supplied

Depletion of high-abundant proteins in body fluids prior to liquid chromatography fourier transform ion cyclotron resonance mass spectrometry.

Today, proteomics is an exciting approach to discover potential biomarkers of different disorders. One challenge with proteomics experiments is the wide concentration range of proteins in various tissues and body fluids. The most abundant component in human body fluids, human serum albumin (HSA), is present at concentrations corresponding to approximately 50% of the total protein content in, e.g., plasma and cerebrospinal fluid (CSF). If this component could be selectively removed, then the chances of observing lower-abundance component of clinical interest would be greatly improved. There are today several approaches of varying specificity available for depletion. In this study, the properties of two commercially available kits, for the removal of HSA and HSA and immunoglobulin G (IgG), respectively, were compared, and the benefits of using depletion steps prior to on-line LC-FTICR MS were evaluated. Both methods were applied on plasma and CSF. To our knowledge, these are the first results reported for CSF. Also, the combination with electrospray LC-FTICR MS is novel. The proportion of depleted HSA and IgG was estimated using global labeling markers for peptide quantification. Both depletion-methods provided a significant reduction of HSA, and the identification of lower abundant components was clearly facilitated. A higher proportion of HSA was removed using the affinity-based removal kit, and consequently more proteins could be identified using this approach.

Adult↗
PubMed2005

Identification of midgut proteins that are differentially expressed in trypanosome-susceptible and normal tsetse flies (Glossina morsitans morsitans).

Molecules in the midgut of tsetse flies (Diptera: Glossinidiae) are thought to play important roles in the life cycle of African trypanosomes by influencing initial parasite establishment and subsequent differentiation events that ultimately lead to maturation of mammal-infective trypanosomes. The molecular composition of the tsetse midgut is, therefore, of critical importance to disease transmission by these medically important vectors. In this study we compared protein expression profiles of midguts of the salmon mutant and wild type Glossina morsitans morsitans Westwood that display marked differences in their susceptibility to infection by African trypanosomes. Isotope coded affinity tag (ICAT) technology was used to identify 207 proteins including 17 that were up regulated and nine that were down regulated in the salmon mutants. Several of the up regulated molecules were previously described as tsetse midgut or salivary gland proteins. Of particular interest was the up regulation in the salmon flies of tsetse midgut EP protein, a recently described molecule with lectin-like activity that was also found to be induced in tsetse by bacterial challenge. The up regulation of the EP protein in midguts of salmon mutants was confirmed by two-dimensional gel electrophoresis and tandem mass spectrometry.

Amino Acid Sequence↗
PubMed2002

Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay.

In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis. However, with the exception of the peroxisome-targeting signal receptors and the receptor docking proteins, the function of most of these proteins, called peroxins, remains largely unknown. One step toward elucidating the function of a protein is to identify its interacting partners. We have used a non-transcription-based bacterial two-hybrid system to analyze the interactions among a set of 12 mammalian peroxins and a yeast protein three-hybrid system to investigate whether proteins that interact with the same peroxin and have overlapping binding sites cooperate or compete for this site. Here we report a detailed interaction map of these peroxins and demonstrate that (i) farnesylation, and not the CAAX motif, of Pex19p strongly enhances its affinity for Pex13p; (ii) the CAAXmotif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11pbeta; and (iii) the C(3)HC(4) RING (really interesting new gene) finger domain of Pex12p does not alter the binding properties of Pex5p for the C-terminal peroxisome-targeting signal PTS1. Finally, we show that the Pex5p-Pex13p interaction is bridged by Pex14p and that the latter molecule exists predominantly as a dimer in vivo. Collectively, as demonstrated in this work with peroxins, these results indicate that the bacterial two-hybrid system is an attractive complementary approach to the conventional transcription-based yeast two-hybrid system for studying protein-protein interactions.

Animals↗
PubMed2025

The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis.

Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus - a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered but retains association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.

Biochemistry and Chemical Biology↗
PubMed2006

Fluorophore-assisted light inactivation of calmodulin involves singlet-oxygen mediated cross-linking and methionine oxidation.

Fluorophore-assisted light inactivation (FALI) permits the targeted inactivation of tagged proteins and, when used with cell-permeable multiuse affinity probes (MAPs), offers important advantages in identifying physiological function, because targeted protein inactivation is possible with spatial and temporal control. However, reliable applications of FALI, also known as chromophore-assisted light inactivation (CALI) with fluorescein derivatives, have been limited by lack of mechanistic information regarding target protein sensitivity. To permit the rational inactivation of targeted proteins, we have identified the oxidizing species and the susceptibility of specific amino acids to modification using the calcium regulatory protein calmodulin (CaM) that, like many essential proteins, regulates signal transduction through the reversible association with a large number of target proteins. Following the covalent and rigid attachment of 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) to helix A, we have identified light-dependent oxidative modifications of endogenous methionines to their corresponding methionine sulfoxides. Initial rates of methionine oxidation correlate with surface accessibility and are insensitive to the distance between the bound fluorophore and individual methionines, which vary between approximately 7 and 40 A. In addition, we observed a loss of histidines, as well as zero-length cross-linking with binding partners corresponding to the CaM-binding sites of smooth myosin light chain kinase and ryanodine receptor. Our results provide a rationale for proteomic screens using FALI to inhibit the function of many signaling proteins, which, like CaM, commonly present methionines at binding interfaces.

Amino Acid Sequence↗
PubMed2003

Prediction of drug metabolism: the case of cytochrome P450 2D6.

Cytochromes P450 (Cyt P450s) constitute the most important biotransformation enzymes involved in the biotransformation of drugs and other xenobiotics. Because drug metabolism by Cyt P450s plays such an important role in the disposition and in the pharmacological and toxicological effects of drugs, early consideration of ADME-properties is increasingly seen as essential for the discovery and the development of new drugs and drug candidates. The primary aim of this paper is to present various computational approaches used to rationalize and predict the activity and substrate selectivity of Cyt P450s, as well as the possibilities and limitations of these approaches, now and in the future. Attention is also paid to the experimental validation of these approaches by using high-throughput screening (HTS) of affinities to drug-drug interactions at the level of Cyt P450-isoenzymes. Since human Cyt P450 2D6 is one of the most important drug metabolizing enzymes and since in this regard much pioneering work has been done with this Cyt P450, Cyt P450 2D6 is chosen as a model for this discussion. Apart from early mechanism-based ab initio calculations on substrates of Cyt P450 2D6, pharmacophore modeling of ligands (i.e. both substrates and inhibitors) of Cyt P450 2D6 and protein homology modeling have been used successfully for the rationalisation and prediction of metabolite formation by this Cyt P450 isoenzyme. Significant protein structure-related species differences have been reported recently. It is concluded that not one computational approach is capable of rationalizing and reliably predicting metabolite formation by Cyt P450 2D6, but that it is rather the combination of the various complimentary approaches. It is moreover concluded, that experimental validation of the computational models and predictions is often still lacking. With the advent of novel, easily and well applicable in vitro based high throughput assays for ligand binding and turnover this limitation could be overcome soon, however. When effective links with other new and recent developments, such as bioinformatics, neural network computing, genomics and proteomics can be created, in silico rationalisation and prediction of drug metabolism by Cyt P450s is likely to become one of the key technologies in early drug discovery and development processes.

Animals↗
PubMed2003

A novel approach for identifying the heme-binding proteins from mouse tissues.

Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of heme-binding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by heme-agarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2-DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

Animals↗
PubMed2002

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes.

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.

CD4 Antigens↗
PubMed2003

Generation of bioreagents for protein chips.

Protein microarrays have the potential to dramatically increase the throughput of proteomic analysis. Protein expression profiling chips with distinct spots of immobilized protein capture agents will allow the simultaneous measurement of hundreds to thousands of proteins from one sample. In contrast to DNA chips, for which the capture probes are easily designed and synthesized, the development of content for protein biochips is a long and laborious process. Careful consideration must be given to the specificities desired, the format of the assay, and the requirements of the capture agents, as well as to process optimization to minimize development time and cost. Monoclonal antibodies have been the prime choice as protein capture agents for the majority of protein chips developed to date. New technologies for the production of protein capture agents are more amenable to automation than traditional monoclonal antibody production and therefore carry the promise for industrialization.

Affinity Labels↗
PubMed2005

Investigating the neuroprotective mechanism of action of a CDK5 inhibitor by phosphoproteome analysis.

Small molecule inhibitors of cyclin-dependent kinase 5 (CDK5) protect neurons from cell death following various insults. To elucidate the cellular mechanism of action we investigated changes in protein phosphorylation in cultured rat cerebellar granule neurons after administration of the CDK5 inhibitor Indolinone A. By immunoblot analysis we detected enhanced phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) and the Jun N-terminal kinase (JNK) substrate c-Jun. Co-administration of U0126, an inhibitor of ERK1/2, or SP600125, an inhibitor of JNK, blocked phosphorylation of ERK1/2 or c-Jun, but did not affect neuroprotection by the CDK5 inhibitor. By metal affinity chromatography, two-dimensional (2D) gel electrophoresis, and MALDI-TOF mass spectrometry we identified several phosphoproteins that accumulated in neurons treated with Indolinone A. Among them were proteins involved in neurotransmitter release, which is consistent with a physiological function of CDK5 in synaptic signaling. Moreover, we identified proteins acting in energy metabolism, protein folding, and oxidative stress response. Similar findings have been reported in yeast following inhibition of Pho85 kinase, which is homologous to mammalian CDK5 and acts in environmental stress signaling. These results suggest that inhibition of CDK5 activates stress responsive proteins that may protect neurons against subsequent injurious stimuli.

Animals↗
PubMed2006

The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032.

The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5.97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2(-) phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5' rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

Bacterial Proteins↗
PubMed2003

Assigning function to yeast proteins by integration of technologies.

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.

Computational Biology↗
PubMed2005

Binding of recombinant PrPc to human plasminogen: kinetic and thermodynamic study using a resonant mirror biosensor.

Transmissible spongiform encephalopathies are a class of sporadic, genetic and transmissible neurodegenerative diseases that affect both humans and animals. Propagation of these diseases is thought to be due to the misfolding of a neuronal glyco-protein, PrP(c), into a pathological insoluble conformer, PrP(Sc). In earlier works, some serum components were identified as exclusive PrP(Sc)-interacting proteins (Fisher et al., Nature 2000;408:479), and thus those macromolecules were thought to represent a potential diagnostic endogenous factor discriminating between normal and pathological prion proteins. In contrast, in agreement with a recent work (Kornblatt et al., Biochem Biophys Res Commun 2003;305:518), in this paper we present a detailed thermodynamic and kinetic characterization of the interaction between recombinant bovine PrP(c 25-242) and the human serum component plasminogen, measured using a resonant mirror technique: our results reveal a high-affinity interaction between the two binding partners. For comparison, the complex obtained from the purified full-length PrP(c) and human plasminogen was also studied: both prion proteins (the recombinant bovine PrP(c 25-242) and the purified full-length PrP(c)) are able to bind human plasminogen. Both kinetic and thermodynamic parameters are affected by the modulation exerted by the H(+) ions in solution. Moreover, the analysis of binding, according to canonical linkage relationships, suggests the involvement of a His residue, consistent with the interaction between other serine (pro)enzymes and their ligands.

Animals↗