Overexpression, purification and characterization of the acidic ribosomal P-proteins from Candida albicans

In all eukaryotic cells, acidic ribosomal P-proteins form a lateral protuberance on the 60S ribosomal subunit-the so-called stalk-structure that plays an important role during protein synthesis. In this work, we report for the first time a full-length cloning of four genes encoding the P-proteins from Candida albicans, their expression in Escherichia coli, purification and characterization of the recombinant proteins. Considerable amino acid sequence similarity was found between the cloned proteins and other known fungal ribosomal P-proteins. On the basis of their phylogenetic relationship and amino acid similarity to their yeast counterparts, the C. albicans P-proteins were named P1A, P1B, P2A and P2B. Using three different approaches, namely: chemical cross-linking method, gel filtration and two-hybrid system, we analyzed mutual interactions among the C. albicans P-proteins. The obtained data showed all the four P-proteins able to form homo-oligomeric complexes. However, the ones found between P1B-P2A and P1A-P2B were dominant forms among the C. albicans P-proteins. Moreover, the strength of interactions between particular proteins was different in these two complexes; the strongest interactions were observed between P1B and P2A proteins, and a significantly weaker one between P1A and P2B proteins.     

Subcellular distribution of the acidic ribosomal P-proteins from Saccharomyces cerevisiae in various environmental conditions

The yeast ribosomal "stalk"--a lateral protuberance on the 60S subunit--consists of four acidic P-proteins, P1A, P1B, P2A and P2B, which play an important role during protein synthesis. Contrary to most ribosomal proteins, which are rapidly degraded in the cytoplasm, P-proteins are found as a cytoplasmic pool and are exchanged with the ribosome-bound proteins during translation. As yet, subcellular trafficking of P-proteins has not been extensively investigated. Therefore, we have characterized--using immunological approaches--the cellular distribution of P-proteins in several environmental conditions, characteristic of yeast cells, such as growth phases, and heat-, osmotic-, and oxygen-stress. Using the western blotting approach, we have shown P-proteins to be present in constant amounts on the ribosomes, despite their exchangeability with the cytoplasmic pool, and regardless of environmental conditions. On the other hand, P-protein level in the cytoplasm decreased sharply throughout the consecutive growth phases, but was not affected by several stress conditions. Applying the electron microscopic technique and immunogold labeling, we have found that P-proteins are located in two cell compartments. The first one is the cytoplasm and the second one--an unexpected place--the cell wall, where P-proteins are fully phosphorylated. Moreover, the existence of P-proteins on the cellular wall is not affected by various environmental conditions.     

IDQD算法预测人类蛋白质相互作用

蛋白质是一切生命活动的物质基础,研究蛋白质的相互作用有助于理解生物过程的分子机制,阐明疾病的分子机理。本文依据蛋白质序列组分特征,应用基于多样性增量的二次判别分析方法,对人类的1 963对蛋白质相互作用进行了预测。自洽检验的各项预测指标均在79%以上,且交叉检验的总精度也大于60%,表明本算法可以用于蛋白质相互作用预测。     

Electrochemical detection of protein-protein interactions using a yeast two hybrid: 17-beta-estradiol as a model

In this work we present a modified yeast two-hybrid bioassay for the highly sensitive detection of protein-protein interactions, based on the electrochemical monitoring of beta-D-galactosidase reporter gene activity, using p-aminophenyl-beta-D-galactopyranoside (PAPG) as a synthetic substrate. In a model system, the sensitive detection of 17-beta-estradiol was achieved at concentrations as low as 10(-11)M (approx 2 pg/ml) by monitoring 17-beta-estradiol receptor dimerization after exposure to 17-beta-estradiol. The sensitivity of this system was higher than that of standard optical methods by three orders of magnitude.     

Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins

Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.     

Mapping protein-protein interactions between MutL and MutH by cross-linking

Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH.MutL complex and to explain the ATP dependence of this interaction.     

Analysis of protein-protein interactions by mutagenesis: direct versus indirect effects

Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information.     

Heat shock protein 90 mediates protein-protein interactions between human aminoacyl-tRNA synthetases

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.     

Analysing protein-protein interactions with the yeast two-hybrid system

Plant research is moving into the post-genomic era. Proteomic-based strategies are now being developed to study functional aspects of the genes predicted from the various genome-sequencing initiatives. All biological processes depend on interactions formed between proteins and the mapping of such interactions on a global scale is providing interesting functional insights. One of the techniques that has proved itself invaluable in the mapping of protein-protein interactions is the yeast two-hybrid system. This system is a sensitive molecular genetic approach for studying protein-protein interactions in vivo. In this review we will introduce the yeast two-hybrid system, discuss modifications of the system that may be of interest to the plant science community and suggest potential applications of the technology.     

Associations between HIV and human pathways revealed by protein-protein interactions and correlated gene expression profiles

Background

AIDS is one of the most devastating diseases in human history. Decades of studies have revealed host factors required for HIV infection, indicating that HIV exploits host processes for its own purposes. HIV infection leads to AIDS as well as various comorbidities. The associations between HIV and human pathways and diseases may reveal non-obvious relationships between HIV and non-HIV-defining diseases.

Principal Findings

Human biological pathways were evaluated and statistically compared against the presence of HIV host factor related genes. All of the obtained scores comparing HIV targeted genes and biological pathways were ranked. Different rank results based on overlapping genes, recovered virus-host interactions, co-expressed genes, and common interactions in human protein-protein interaction networks were obtained. Correlations between rankings suggested that these measures yielded diverse rankings. Rank combination of these ranks led to a final ranking of HIV-associated pathways, which revealed that HIV is associated with immune cell-related pathways and several cancer-related pathways. The proposed method is also applicable to the evaluation of associations between other pathogens and human pathways and diseases.

Conclusions

Our results suggest that HIV infection shares common molecular mechanisms with certain signaling pathways and cancers. Interference in apoptosis pathways and the long-term suppression of immune system functions by HIV infection might contribute to tumorigenesis. Relationships between HIV infection and human pathways of disease may aid in the identification of common drug targets for viral infections and other diseases.     

Similarity between protein-protein and protein-carbohydrate interactions, revealed by two crystal structures of lectins from the roots of pokeweed

The roots of pokeweed (Phytolacca americana) are known to contain the lectins designated PL-A, PL-B, PL-C, PL-D1, and PL-D2. Of these lectins, the crystal structures of two PLs, the ligand-free PL-C and the complex of PL-D2 with tri-N-acetylchitotriose, have been determined at 1.8A resolution. The polypeptide chains of PL-C and PL-D2 form three and two repetitive chitin-binding domains, respectively. In the crystal structure of the PL-D2 complex, one trisaccharide molecule is shared mainly between two neighboring molecules related to each other by a crystallographic 2(1)-screw axis, and infinite helical chains of complexed molecules are generated by the sharing of ligand molecules. The crystal structure of PL-C reveals that the molecule is a dimer of two identical subunits, whose polypeptide chains are located in a head-to-tail fashion by a molecular 2-fold axis. Three putative carbohydrate-binding sites in each subunit are located in the dimer interface. The dimerization of PL-C is performed through the hydrophobic interactions between the carbohydrate-binding sites of the opposite domains in the dimer, leading to a distinct dimerization mode from that of wheat-germ agglutinin. Three aromatic residues in each carbohydrate-binding site of PL-C are involved in the dimerization. These residues correspond to the residues that interact mainly with the trisaccharide in the PL-D2 complex and appear to mimic the saccharide residues in the complex. Consequently, the present structure of the PL-C dimer has no room for accommodating carbohydrate. The quaternary structure of PL-C formed through these putative carbohydrate-binding residues may lead to the lack of hemagglutinating activity.     

Hot spot prediction in protein-protein interactions by an ensemble system

Background

Hot spot residues are functional sites in protein interaction interfaces. The identification of hot spot residues is time-consuming and laborious using experimental methods. In order to address the issue, many computational methods have been developed to predict hot spot residues. Moreover, most prediction methods are based on structural features, sequence characteristics, and/or other protein features.

Results

This paper proposed an ensemble learning method to predict hot spot residues that only uses sequence features and the relative accessible surface area of amino acid sequences. In this work, a novel feature selection technique was developed, an auto-correlation function combined with a sliding window technique was applied to obtain the characteristics of amino acid residues in protein sequence, and an ensemble classifier with SVM and KNN base classifiers was built to achieve the best classification performance.

Conclusion

The experimental results showed that our model yields the highest F1 score of 0.92 and an MCC value of 0.87 on ASEdb dataset. Compared with other machine learning methods, our model achieves a big improvement in hot spot prediction.

Availability

http://deeplearner.ahu.edu.cn/web/HotspotEL.htm.

     

Single-molecule observation of protein-protein interactions in the chaperonin system

We have analyzed the dynamics of the chaperonin (GroEL)-cochaperonin (GroES) interaction at the single-molecule level. In the presence of ATP and non-native protein, binding of GroES to the immobilized GroEL occurred at a rate that is consistent with bulk kinetics measurements. However, the release of GroES from GroEL occurred after a lag period ( approximately 3 s) that was not recognized in earlier bulk-phase studies. This observation suggests a new kinetic intermediate in the GroEL-GroES reaction pathway.     

Prediction of protein-protein interactions: the CAPRI experiment, its evaluation and implications

Given the increasing interest in protein-protein interactions, the prediction of these interactions from sequence and structural information has become a booming activity. CAPRI, the community-wide experiment for assessing blind predictions of protein-protein interactions, is playing an important role in fostering progress in docking procedures. At the same time, novel methods are being derived for predicting regions of a protein that are likely to interact and for characterizing putative intermolecular contacts from sequence and structural data. Together with docking procedures, these methods provide an integrated computational approach that should be a valuable complement to genome-scale experimental studies of protein-protein interactions.     

Molecular interactions between multihaem cytochromes: probing the protein-protein interactions between pentahaem cytochromes of a nitrite reductase complex

The cytochrome c nitrite reductase NrfA is a 53?kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21?kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50?nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.     

Probabilistic prediction and ranking of human protein-protein interactions

Background  

Although the prediction of protein-protein interactions has been extensively investigated for yeast, few such datasets exist for the far larger proteome in human. Furthermore, it has recently been estimated that the overall average false positive rate of available computational and high-throughput experimental interaction datasets is as high as 90%.     

Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.     

Analysis of sequence-reactivity space for protein-protein interactions

Sequence–reactivity space is defined by the relationships between amino acid type choices at some residue positions in a protein and the reactivities of the resulting variants. We are studying Kazal superfamily serine proteinase inhibitors, under substitution of any combination of residue types at 10 binding‐region positions. Reactivities are defined by the standard free energy of association for an inhibitor against an enzyme, and we are interested in both the strength (the free energy value) and specificity (relative free energy values for one inhibitor against different enzymes). Characterizing the structure of such a space poses several interesting questions: (1) How many sequences achieve particular strength and specificity characteristics? (2) What is the best such sequence? (3) What are some nearly‐as‐good alternatives? (4) What are their common residue type characteristics (e.g., conservation and correlation)? Although these problems are all highly combinatorial in nature, this article develops an efficient, integrated mechanism to address them under a data‐driven model that predicts reactivity for given sequences. We employ sampling and a novel deterministic distribution propagation algorithm, in order to determine both the reactivity distribution and sequence composition statistics; integer programming and a novel branch‐and‐bound search algorithm, in order to optimize sequences and enumerate near‐optimal sequences; and correlation‐based sequence decomposition, in order to identify sequence motifs. We demonstrate the value of our mechanism in analyzing the Kazal superfamily sequence–reactivity space, providing insights into the underlying biochemistry and suggesting hypotheses for further experimental consideration. In general, our mechanism offers a valuable tool for investigating the available degrees of freedom in protein design within a combined computational–experimental framework. Proteins 2005. © 2004 Wiley‐Liss, Inc.     

Reconstitution of fibroblast growth factor receptor interactions in the yeast two hybrid system

Fibroblast growth factors (FGF) activate their receptors through the formation of trimolecular complexes, composed of a ligand, a receptor, and a heparan sulfate oligosaccharide, all of which are members of particularly large families capable of multiple interactions in a combinatorial fashion. Understanding this large network of interactions not only presents a great challenge, but is practically beyond the capacity of most classical techniques routinely used to study ligand receptor interactions. We have used the yeast two hybrid system to study protein-protein interaction in the FGF family. Both ligand and receptor ectodomains are properly folded and functional in the yeast. Basic FGF (bFGF) expressed in the yeast dimerizes spontaneously. This self-assembly occurs at low affinity, which can be greatly enhanced by the introduction of heparin, supporting a defined role for heparin in bFGF dimerization. Screening a rat embryo cDNA library with bFGF in the yeast two hybrid system identified a short variant of FGF receptor 1, found most frequently in embryonal and tumor cells and which possesses affinity toward bFGF that is significantly greater than that of the more abundant, full-length receptor. We find the yeast two hybrid system, a most suitable alternative method for the analysis of growth factor-receptor interactions as well as for screening for novel interacting proteins and modulators of FGF and its receptors.     

Localization of protein-protein interactions between subunits of phytochrome.

We have used a novel assay based on protein fusions with lambda repressor to identify two small regions within phytochrome's carboxy-terminal domain that are capable of mediating dimerization. Using an in vivo assay, fusions between the DNA binding, amino-terminal domain of lambda repressor and fragments from oat PhyA phytochrome have been assayed for increased repressor activity, an indicator of dimerization. In this assay system, regions of oat phytochrome between amino acids V623-S673 and N1049-Q1129 have been shown to increase repressor activity. These short spans are highly conserved between proteins belonging to the phytochrome PhyA family. Embedded within these sequences are four segments that could potentially form amphipathic alpha helices. Two of the segments are well conserved between PhyA phytochrome and phytochromes encoded by the phyB and phyC genes, suggesting that heterodimers might form by way of subunit interaction at these sites.