Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli.     

Mass spectrometric identification of RNA binding proteins from dried EMSA gels

Electrophoretic mobility shift assays (EMSA) are commonly employed for the analysis of nucleic acid/ protein interactions with a native gel system. Here, we report a method to identify RNA binding proteins from a dried EMSA gel by mass spectrometry following autoradiography. Compared to wet gel exposure, our approach resulted in an improved protein identification sensitivity and RNA/protein complex isolation accuracy. The method described here is useful for the large scale characterization of RNA- or DNA-protein complexes.     

The RNA binding domains of the nuclear poly(A)-binding protein

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.     

Identification and characterization of proteins that interact with the carboxy terminus of poly(A)-binding protein and inhibit translation in vitro

Poly(A)-binding proteins (PABPs) are multifunctional proteins that play important roles in mRNA stability and protein translation. Two cucumber ( Cucumis sativus L.) proteins, PCI6 (PABP-CT-interacting) and PCI243 were identified based on ability to interact with the carboxy terminus (CT) of PABP in yeast two-hybrid and in vitro binding assays. PCI6 and PCI243 share a conserved amino acid domain (SxLnpnApxFxP) in common with human PABP-CT interactors, and with Arabidopsis ERD15 (early-responsive to dehydration). Deletion analysis and point mutations indicate that presence of this domain is necessary for the interaction, and tests with ERD15 demonstrate that it is predictive of interaction. Other plant proteins possessing this domain fall into two categories: small, acidic proteins like PCI6, PCI243 and ERD15, and larger neutral proteins that also include an RNA recognition motif. PCI6 is expressed in a range of tissues, e.g., leaves, roots, stems and flowers, and follows a diurnal pattern of expression, increasing during light hours and declining overnight. In wheat germ and mouse ascites Krebs-2 in vitro translation systems, PCI6 inhibited translation whereas the non-interacting mutant, PCI6-23A, did not or had a greatly reduced effect. The activity of PCI6, therefore, is reminiscent of that of human PABP-interacting protein 2 (Paip2). These results demonstrate a novel interaction between PABP and several plant proteins sharing a SxLnpxApxFxP motif, with possible implications for translational regulation.     

Identification and characterization of proteins that selectively interact with isoforms of the mRNA binding protein AUF1 (hnRNP D)

The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs. AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation. Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1. We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1. Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains.

A family of eukaryotic RNA binding proteins is defined by the conserved RNP motif. The poly(A) binding protein has four such motifs. We report on the isolation and structural characterization of several variant cDNA clones, as well as of a gene encoding this protein in Xenopus laevis embryos. Wild-type protein as well as truncated versions carrying isolated single motifs or artificial combinations of two and more such elements were characterized for their ability to bind specifically to RNA homopolymers. Three of the isolated repeats were functional in specific RNA binding, whereas the N-terminal RNP motif was non-functional. Combinatorial effects in RNA binding were measured with constructs carrying multiple repeats, which were not predictable from the activity of isolated domains.     

Identification of Arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein

Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement. The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins. Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants, and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7.     

Structure and function of poly(A) binding proteins

Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.     

Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA

During polyadenylation of mRNA precursors in metazoan cells, poly(A) polymerase is stimulated by the nuclear poly(A) binding protein PABPN1. We report that stimulation depends on binding of PABPN1 to the substrate RNA directly adjacent to poly(A) polymerase and results in an approximately 80-fold increase in the apparent affinity of poly(A) polymerase for RNA without significant effect on catalytic efficiency. PABPN1 associates directly with poly(A) polymerase either upon allosteric activation by oligo(A) or, in the absence of RNA, upon deletion of its N-terminal domain. The N-terminal domain of PABPN1 may function to inhibit undesirable interactions of the protein; the inhibition is relieved upon RNA binding. Tethering of poly(A) polymerase is mediated largely by the C-terminal domain of PABPN1 and is necessary but not sufficient for stimulation of the enzyme; an additional interaction dependent on a coiled-coil structure located within the N-terminal domain of PABPN1 is required for a productive interaction.     

Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly

Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions. Proteins Suppl. 2:63–73, 1998. © 1998 Wiley-Liss, Inc.     

CHK1相互作用蛋白的筛选

将CHK1基因克隆入酵母双杂交载体中,转化入酵母菌AH109,将转化有CHK1基因的酵母菌AH109培养并再转化人前列腺cDNA库质粒,检测报告基因的表达情况,筛选与CHK1相互作用的蛋白。共转化子中有4个β-半乳糖苷酶活性分析和α-半乳糖苷酶活性分析结果为阳性的克隆,但测序结果其中有2个克隆为相同序列。本实验筛选到3个与CHK1相互作用的蛋白,此结果为进一步研究与CHK1相互作用的蛋白奠定了基础。     

Conformity of RNAs that interact with tetranucleotide loop binding proteins.

A group of RNA binding proteins, termed tetraloop binding proteins, includes ribosomal protein S15 and protein SRP19 of signal recognition particle. They are primary RNA binding proteins, recognize RNA tetranucleotide loops with a GNAR consensus motif, and require a helical region located adjacent to the tetraloop. Closely related RNA structures that fit these criteria appear in helix 6 of SRP RNA, in helices 22 and 23A of 16 S ribosomal RNA, and, as a pseudoknot, in the regulatory region of the rpsO gene.     

Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition

Mitochondrial membrane permeabilization is a rate-limiting step of cell death. This process is, at least in part, mediated by opening of the permeability transition pore complex (PTPC) Several soluble proteins from the mitochondrial intermembrane space and matrix are involved in the activation of catabolic hydrolases including caspases and nucleases. We therefore investigated the composition of a mixture of proteins released from purified mitochondria upon PTPC opening. This mixture was subjected to a novel proteomics/mass spectrometric approach designed to identify a maximum of peptides. Peptides from a total of 79 known proteins or genes were identified. In addition, 21 matches with expressed sequence tags (EST) were obtained. Among the known proteins, several may have indirect or direct pro-apoptotic properties. Thus endozepine, a ligand of the peripheral benzodiazepin receptor (whose occupation may facilitate mitochondrial membrane permeabilization), was found among the released proteins. Several proteins involved in protein import were also released, namely the so-called X-linked deafness dystonia protein (DDP) and the glucose regulated protein 75 (grb75), meaning that protein import may become irreversibly disrupted in mitochondria of apoptotic cells. In addition, a number of catabolic enzymes are detected: arginase 1 (which degrades arginine), sulfite oxidase (which degrades sulfur amino acids), and epoxide hydrolase. Although the functional impact of each of these proteins on apoptosis remains elusive, the present data bank of mitochondrial proteins released upon PTPC opening should help further elucidation of the death process.     

Xenopus embryonic poly(A) binding protein 2 (ePABP2) defines a new family of cytoplasmic poly(A) binding proteins expressed during the early stages of vertebrate development

We describe a new RNA binding protein from Xenopus we have named ePABP2 (embryonic poly(A) binding protein type II). Based on amino acid similarity, ePABP2 is closely related to the ubiquitously expressed nuclear PABP2 protein that directs the elongation of mRNA poly(A) tails during pre-mRNA processing. However, in contrast to known PABP2 proteins, Xenopus ePABP2 is a cytoplasmic protein that is predominantly expressed during the early stages of Xenopus development and in adult ovarian tissue. Biochemical experiments indicate ePABP2 binds poly(A) with specificity and that this binding requires the RRM domain. Mouse and human ePABP2 proteins were also identified and mouse ePABP2 expression is also confined to the earliest stages of mouse development and adult ovarian tissue. We propose that Xenopus ePABP2 is the founding member of a new class of poly(A) binding proteins expressed in vertebrate embryos. Possible roles for this protein in regulating mRNA function in early vertebrate development are discussed.     

Disulfide bond formation between RNA binding domains is used to regulate mRNA binding activity of the chloroplast poly(A)-binding protein

Binding of the chloroplast poly(A)-binding protein, RB47, to the psbA mRNA is regulated in response to light and is required for translation of this mRNA in chloroplasts. The RNA binding activity of RB47 can be modulated in vitro by oxidation and reduction. Site-directed mutations to individual cysteine residues in each of the four RNA binding domains of RB47 showed that changing single cysteines to serines in domains 2 or 3 reduced, but did not eliminate, the ability of RB47 to be redox-regulated. Simultaneously changing cysteines to serines in both domains 2 and 3 resulted in the production of RB47 protein that was insensitive to redox regulation but retained the ability to bind the psbA mRNA at high affinity. The poly(A)-binding protein from Saccharomyces cerevisiae lacks cysteine residues in RNA binding domains 2 and 3, and this poly(A)-binding protein lacks the ability to be regulated by oxidation or reduction. These data show that disulfide bond formation between RNA binding domains in a poly(A)-binding protein can be used to regulate the ability of this protein to bind mRNA and suggest that redox regulation of RNA binding activity may be used to regulate translation in organisms whose poly(A)-binding proteins contain these critical cysteine residues.     

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical ππ-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). All interactions are validated by mutational analyses and the high-resolution structures presented here will guide further studies to understand how both proteins act together in cellular processes.     

Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.