A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A putative spermidine synthase interacts with flagellar switch protein FliM and regulates motility in Helicobacter pylori

The flagellar motor is an important virulence factor in infection by many bacterial pathogens. Motor function can be modulated by chemotactic proteins and recently appreciated proteins that are not part of the flagellar or chemotaxis systems. How these latter proteins affect flagellar activity is not fully understood. Here, we identified spermidine synthase SpeE as an interacting partner of switch protein FliM in Helicobacter pylori using pull‐down assay and mass spectrometry. To understand how SpeE contributes to flagellar motility, a speE‐null mutant was generated and its motility behavior was evaluated. We found that deletion of SpeE did not affect flagellar formation, but induced clockwise rotation bias. We further determined the crystal structure of the FliM‐SpeE complex at 2.7 Å resolution. SpeE dimer binds to FliM with micromolar binding affinity, and their interaction is mediated through the β1' and β2' region of FliM middle domain. The FliM‐SpeE binding interface partially overlaps with the FliM surface that interacts with FliG and is essential for proper flagellar rotational switching. By a combination of protein sequence conservation analysis and pull‐down assays using FliM and SpeE orthologues in E. coli, our data suggest that FliM‐SpeE association is unique to Helicobacter species.     

Isolation and characterisation of cDNA encoding a wheat heavy metal‐associated isoprenylated protein involved in stress responses

In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal‐associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy‐terminal end. To investigate the roles of wheat heavy metal‐associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal‐associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real‐time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up‐regulated by ABA exposure or wounding. Additionally, TaHIPP1 over‐expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu²⁺ and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1‐GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus‐induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses.     

Characterization of G3BPs: Tissue specific expression,chromosomal localisation and rasGAP120 binding studies

The G3BP (ras‐GTPase‐Activating Protein SH3‐Domain‐Binding Protein) family of proteins has been implicated in both signal transduction and RNA‐metabolism. We have previously identified human G3BP‐1, G3BP‐2, and mouse G3BP‐2. Here, we report the cloning of mouse G3BP‐1, the discovery of two alternatively spliced isoforms of mouse, and human G3BP‐2 (G3BP‐2a and G3BP‐2b), and the chromosomal localisation of human G3BP‐1 and G3BP‐2, which map to 5q14.2‐5q33.3 and 4q12‐4q24 respectively. We mapped the rasGAP¹²⁰ interactive region of the G3BP‐2 isoforms and show that both G3BP‐2a and G3BP‐2b use an N‐terminal NTF2‐like domain for rasGAP¹²⁰ binding rather than several available proline‐rich (PxxP) motifs found in members of the G3BPs. Furthermore, we have characterized the protein expression of both G3BP‐1 and G3BP‐2a/b in adult mouse tissues, and show them to be both tissue and isoform specific. J. Cell. Biochem. 84: 173–187, 2002. © 2001 Wiley‐Liss, Inc.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

Despite the great interest in identifying protein–protein interactions (PPIs) in biological systems, only a few attempts have been made at large‐scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs in vivo at subcellular resolution. However, when the non‐fluorescent fragments are highly expressed, spontaneous and irreversible self‐assembly of the split halves can easily generate false positives. The recently developed tripartite split‐GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β‐estradiol‐inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split‐GFP association in plant cells and affirm that the tripartite split‐GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split‐GFP association and dual‐intein‐mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof‐of‐concept implementation of the tripartite split‐GFP system as a potential tool for membrane PPI screens in planta.     

Characterization of a mitochondrially targeted single-stranded DNA-binding protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.     

Calmodulin‐related CML24 interacts with ATG4b and affects autophagy progression in Arabidopsis

Plants encounter environmental stress challenges that are distinct from those of other eukaryotes because of their relative immobility. Therefore, plants may have evolved distinct regulatory mechanisms for conserved cellular functions. Plants, like other eukaryotes, share aspects of both calcium‐ and calmodulin‐based cellular signaling and the autophagic process of cellular renewal. Here, we report a novel function for an Arabidopsis calmodulin‐related protein, CML24, and insight into ATG4‐regulated autophagy. CML24 interacts with ATG4b in yeast two‐hybrid, in vitro pull‐down and transient tobacco cell transformation assays. Mutants with missense mutations in CML24 have aberrant ATG4 activity patterns in in vitro extract assays, altered ATG8 accumulation levels, an altered pattern of GFP–ATG8‐decorated cellular structures, and altered recovery from darkness‐induced starvation. Together, these results support the conclusion that CML24 affects autophagy progression through interactions with ATG4.     

Cryoprotective mechanism of a small intrinsically disordered dehydrin protein

Dehydration proteins (Dehydrins) are expressed during dehydration stress in plants and are thought to protect plant proteins and membranes from the loss of water during drought and at cold temperatures. Several different dehydrins have been shown to protect lactate dehydrogenase (LDH) from damage from being frozen and thawed. We show here that a 48 residue K₂ dehydrin from Vitis riparia protects LDH more effectively than bovine serum albumin, a protein with known cryoprotective function. Light scattering and 8‐anilino‐1‐naphthalene sulfonate fluorescence experiments show that dehydrins prevent aggregation and unfolding of the enzyme. The cryoprotective effects of LDH are reduced by the addition of salt, suggesting that the positively charged K‐segments are attracted to a negatively charged surface but this does not result in binding. Overall K₂ is an intrinsically disordered protein; nuclear magnetic resonance relaxation experiments indicate that the two‐terminal, Lys‐rich K‐segments show a weak propensity for α‐helicity and are flexible, and that the central, polar rich phi‐segment has no secondary structure preference and is highly flexible. We propose that the phi‐segments in dehydrins are important for maintaining the disordered structure so that the protein can act as a molecular shield to prevent partially denatured proteins from interacting with one another, whereas the K‐segments may help to localize the dehydrin near the enzyme surface.     

Modification of PATase by L/F‐transferase generates a ClpS‐dependent N‐end rule substrate in Escherichia coli

The N‐end rule pathway is conserved from bacteria to man and determines the half‐life of a protein based on its N‐terminal amino acid. In Escherichia coli, model substrates bearing an N‐degron are recognised by ClpS and degraded by ClpAP in an ATP‐dependent manner. Here, we report the isolation of 23 ClpS‐interacting proteins from E. coli. Our data show that at least one of these interacting proteins—putrescine aminotransferase (PATase)—is post‐translationally modified to generate a primary N‐degron. Remarkably, the N‐terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl‐tRNA‐protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N‐terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N‐end rule pathway, through the ClpAPS‐mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N‐degron, which is required for substrate delivery to ClpA.     

A putative Toll/interleukin‐1 receptor domain protein from Helicobacter pylori is dimeric in solution and interacts with human Toll‐like receptor adaptor myeloid differentiation primary response 88

Helicobacter pylori, an important human pathogen, is capable of causing persistent infection with minimal immune response. The first line of defense during H. pylori infection is through gastric epithelial cells that present TLR, A family of bacterial proteins that share homology with the Toll/IL‐1 receptor (TIR) domain were identified. Bacterial TIR proteins (BTP) mimic human TIR domain proteins and act on myeloid differentiation primary response gene 88 (MyD88) signaling pathways to suppress TLR signaling. H. pylori may also produce a similar protein. A putative H. pylori BTP was found based on sequence homology. The corresponding gene hp1437 was inserted into an expression vector in fusion with an N‐terminal cleavable 6his‐tag. The recombinant protein, 6his‐HP1437, was purified using nickel affinity chromatography with a yield of 8 mg/L culture. Oligomerization of HP1437 was investigated by size‐exclusion chromatography. It was found that HP1437 forms dimers in solution similar to other BTPs. Furthermore, glutathione S‐transferase pull down assays identified an interaction between HP1437 and human TIR domain adaptor MyD88. These findings suggest that HP1437 has the characteristic features of BTPs and may play a direct role in reducing immune response against H. pylori by binding to MyD88 and pave the way for an in‐depth characterization of this putative novel H. pylori virulence factor.     

Single‐step protease cleavage elution for identification of protein–protein interactions from GST pull‐down and mass spectrometry

The study of protein–protein interactions is a major theme in biological disciplines. Pull‐down or affinity‐precipitation assays using GST fusion proteins have become one of the most common and valuable approaches to identify novel binding partners for proteins of interest (bait). Non‐specific binding of prey proteins to the beads or to GST itself, however, inevitably complicates and impedes subsequent analysis of pull‐down results. A variety of measures, each with inherent advantages and limitations, can minimise the extent of the background. This technical brief details and tests a modification of established GST pull‐down protocols. By specifically eluting only the bait (minus the GST tag) and the associated non‐specific binding proteins with a simple, single‐step protease cleavage, a cleaner platform for downstream protein identification with MS is established. We present a proof of concept for this method, as evidenced by a GST pull‐down/MS case study of the small guanosine triphosphatase (GTPase) Rab31 in which: (i) sensitivity was enhanced, (ii) a reduced level of background was observed, (iii) distinguishability of non‐specific contaminant proteins from genuine binders was improved and (iv) a putative new protein–protein interaction was discovered. Our protease cleavage step is readily applicable to all further affinity tag pull‐downs.     

A novel signal transduction protein: Combination of solute binding and tandem PAS‐like sensor domains in one polypeptide chain

We report the structural and biochemical characterization of a novel periplasmic ligand‐binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N‐terminal and a tandem PAS‐like sensor domain at the C‐terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS‐like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS‐like domain of the tandem PAS‐like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS‐like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non‐redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.     

Efficient identification of tubby‐binding proteins by an improved system of T7 phage display

Mutation in the tubby gene causes adult‐onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby‐like protein 1 (Tulp1), whose C‐terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N‐terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N‐terminus (tubby‐N) as bait to identify unknown binding proteins with open‐reading‐frame (ORF) phage display. T7 phage display was engineered with three improvements: high‐quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait‐binding proteins in as fast as ～4–7 days. While phage display with conventional cDNA libraries identifies high percentage of out‐of‐frame unnatural short peptides, all 28 tubby‐N‐binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two‐hybrid assay and protein pull‐down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby‐specific binding protein. These data suggest that tubby‐N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly‐engineered ORF phage display is a powerful technology to identify unknown protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.     

Discovery of binding proteins for a protein target using protein–protein docking‐based virtual screening

Target structure‐based virtual screening, which employs protein‐small molecule docking to identify potential ligands, has been widely used in small‐molecule drug discovery. In the present study, we used a protein–protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all‐to‐all protein–protein docking run on a large dataset was performed. The three‐dimensional rigid docking program SDOCK was used to examine protein–protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z‐score, and convergency of the low‐score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all‐to‐all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor‐α (TNFα), which is a well‐known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top‐ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein–protein docking for the discovery of novel binding proteins for specific protein targets. Proteins 2014; 82:2472–2482. © 2014 Wiley Periodicals, Inc.     

The Ca2+‐binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer

Plasma membrane‐associated Ca²⁺‐binding protein–2 (PCaP2) of Arabidopsis thaliana is a novel‐type protein that binds to the Ca²⁺/calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca²⁺. Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N–terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca²⁺/calmodulin complex, and that the residual domain of PCaP2 binds to free Ca²⁺. In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild‐type. To examine the function of each domain in root hair cells, we over‐expressed PCaP2 and its variants using the root hair cell‐specific EXPANSIN A7 promoter. Transgenic lines over‐expressing PCaP2, PCaP2^G2A (second glycine substituted by alanine) and ?23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over‐expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP‐tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca²⁺ and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.