Revisiting Date and Party Hubs: Novel Approaches to Role Assignment in Protein Interaction Networks

The idea of 'date' and 'party' hubs has been influential in the study of protein-protein interaction networks. Date hubs display low co-expression with their partners, whilst party hubs have high co-expression. It was proposed that party hubs are local coordinators whereas date hubs are global connectors. Here we show that the reported importance of date hubs to network connectivity can in fact be attributed to a tiny subset of them. Crucially, these few, extremely central, hubs do not display particularly low expression correlation, undermining the idea of a link between this quantity and hub function. The date/party distinction was originally motivated by an approximately bimodal distribution of hub co-expression; we show that this feature is not always robust to methodological changes. Additionally, topological properties of hubs do not in general correlate with co-expression. Thus, we suggest that a date/party dichotomy is not meaningful and it might be more useful to conceive of roles for protein-protein interactions rather than individual proteins. We find significant correlations between interaction centrality and the functional similarity of the interacting proteins.Comment: 27 pages, 5 main figures, 4 supplementary figure

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, the RESPARI Pasteur Network

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m7 guanosine cap

In eukaryotes the majority of mRNAs have an m7G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure

Genomes 2000 International Conference on Microbial and Model Genomes

On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine

Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2-/- mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this 'stress' keratin is regulated

Hubs with Network Motifs Organize Modularity Dynamically in the Protein-Protein Interaction Network of Yeast

BACKGROUND: It has been recognized that modular organization pervades biological complexity. Based on network analysis, 'party hubs' and 'date hubs' were proposed to understand the basic principle of module organization of biomolecular networks. However, recent study on hubs has suggested that there is no clear evidence for coexistence of 'party hubs' and 'date hubs'. Thus, an open question has been raised as to whether or not 'party hubs' and 'date hubs' truly exist in yeast interactome. METHODOLOGY: In contrast to previous studies focusing on the partners of a hub or the individual proteins around the hub, our work aims to study the network motifs of a hub or interactions among individual proteins including the hub and its neighbors. Depending on the relationship between a hub's network motifs and protein complexes, we define two new types of hubs, 'motif party hubs' and 'motif date hubs', which have the same characteristics as the original 'party hubs' and 'date hubs' respectively. The network motifs of these two types of hubs display significantly different features in spatial distribution (or cellular localizations), co-expression in microarray data, controlling topological structure of network, and organizing modularity. CONCLUSION: By virtue of network motifs, we basically solved the open question about 'party hubs' and 'date hubs' which was raised by previous studies. Specifically, at the level of network motifs instead of individual proteins, we found two types of hubs, motif party hubs (mPHs) and motif date hubs (mDHs), whose network motifs display distinct characteristics on biological functions. In addition, in this paper we studied network motifs from a different viewpoint. That is, we show that a network motif should not be merely considered as an interaction pattern but be considered as an essential function unit in organizing modules of networks

RNA Captor: A Tool for RNA Characterization

Background: In the genome era, characterizing the structure and the function of RNA molecules remains a major challenge. Alternative transcripts and non-protein-coding genes are poorly recognized by the current genome-annotation algorithms and efficient tools are needed to isolate the less-abundant or stable RNAs. Results: A universal RNA-tagging method using the T4 RNA ligase 2 and special adapters is reported. Based on this system, protocols for RACE PCR and full-length cDNA library construction have been developed. The RNA tagging conditions were thoroughly optimized and compared to previous methods by using a biochemical oligonucleotide tagging assay and RACE PCRs on a range of transcripts. In addition, two large-scale full-length cDNA inventories relying on this method are presented. Conclusion: The RNA Captor is a straightforward and accessible protocol. The sensitivity of this approach was shown to be higher compared to previous methods, and applicable on messenger RNAs, non-protein-coding RNAs, transcription-start sites and microRNA-directed cleavage sites of transcripts. This strategy could also be used to study other classes of RNA and in deep sequencing experiments

Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage

© 2012 Millet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.This work was supported by the Research Grant Council of Hong Kong (RGC#760208)and the RESPARI project of the International Network of Pasteur Institutes

Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis

Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5′-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein–protein interaction with the cellular translation machinery

Using Ribosomal Protein Genes as Reference: A Tale of Caution

Background: Housekeeping genes are needed in every tissue as their expression is required for survival, integrity or duplication of every cell. Housekeeping genes commonly have been used as reference genes to normalize gene expression data, the underlying assumption being that they are expressed in every cell type at approximately the same level. Often, the terms "reference genes'' and "housekeeping genes'' are used interchangeably. In this paper, we would like to distinguish between these terms. Consensus is growing that housekeeping genes which have traditionally been used to normalize gene expression data are not good reference genes. Recently, ribosomal protein genes have been suggested as reference genes based on a meta-analysis of publicly available microarray data. Methodology/Principal Findings: We have applied several statistical tools on a dataset of 70 microarrays representing 22 different tissues, to assess and visualize expression stability of ribosomal protein genes. We confirmed the housekeeping status of these genes, but further estimated expression stability across tissues in order to assess their potential as reference genes. One- and two-way ANOVA revealed that all ribosomal protein genes have significant expression variation across tissues and exhibit tissue-dependent expression behavior as a group. Via multidimensional unfolding analysis, we visualized this tissue-dependency. In addition, we explored mechanisms that may cause tissue dependent effects of individual ribosomal protein genes. Conclusions/Significance: Here we provide statistical and biological evidence that ribosomal protein genes exhibit important tissue-dependent variation in mRNA expression. Though these genes are most stably expressed of all investigated genes in a meta-analysis they cannot be considered true reference genes