A Systemic Receptor Network Triggered by Human cytomegalovirus Entry

Virus entry is a multistep process that triggers a variety of cellular pathways interconnecting into a complex network, yet the molecular complexity of this network remains largely unsolved. Here, by employing systems biology approach, we reveal a systemic virus-entry network initiated by human cytomegalovirus (HCMV), a widespread opportunistic pathogen. This network contains all known interactions and functional modules (i.e. groups of proteins) coordinately responding to HCMV entry. The number of both genes and functional modules activated in this network dramatically declines shortly, within 25 min post-infection. While modules annotated as receptor system, ion transport, and immune response are continuously activated during the entire process of HCMV entry, those for cell adhesion and skeletal movement are specifically activated during viral early attachment, and those for immune response during virus entry. HCMV entry requires a complex receptor network involving different cellular components, comprising not only cell surface receptors, but also pathway components in signal transduction, skeletal development, immune response, endocytosis, ion transport, macromolecule metabolism and chromatin remodeling. Interestingly, genes that function in chromatin remodeling are the most abundant in this receptor system, suggesting that global modulation of transcriptions is one of the most important events in HCMV entry. Results of in silico knock out further reveal that this entire receptor network is primarily controlled by multiple elements, such as EGFR (Epidermal Growth Factor) and SLC10A1 (sodium/bile acid cotransporter family, member 1). Thus, our results demonstrate that a complex systemic network, in which components coordinating efficiently in time and space contributes to virus entry.Comment: 26 page

Analysis of the role of the C-terminal tail in the regulation of the epidermal growth factor receptor

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer

Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

In vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms "PI3K-Akt", "transforming growth factor-β receptor", "ErbB", and "HIF-1" from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.Peer reviewe

Characterizing the Interplay between FAK, Src, and ACK2 in the Regulation of SH3PX1 Activity

Sorting nexin, SH3PX1, has been associated with endocytosis through its interactions with key endocytic proteins involved in the processing of cell surface receptors. These dramatic cellular effects, largely associated with the tyrosine phosphorylation of SH3PX1, have elicited interest in determining the molecular mechanisms underlying this phosphorylation event. The first major focus of these studies was to develop reagents that could be used to further characterize the role of SH3PX1 in endocytosis. SH3PX1 has been identified as a substrate of the nonreceptor tyrosine kinase, activated Cdc42-associated kinase-2 (ACK2), and the resulting phosphorylation enhances degradation of the epidermal growth factor (EGF) receptor in cells. In order to further characterize ACK2-catalyzed SH3PX1 phosphorylation, we have identified tyrosine 287 as a major site of phosphorylation by mass spectrometry. Moreover, we have shown that the pyridopyrimidine PD158780 is a potent inhibitor of ACK2 kinase activity in vitro (IC50 ~80 pM). Together, we believe that phosphorylation-defective mutants of SH3PX1 and small molecule inhibitors of ACK2 kinase activity will help to further establish the roles of ACK2 and SH3PX1 in EGF receptor processing. The second aspect of these studies involved the identification and characterization of novel tyrosine kinases that phosphorylate SH3PX1 in cells, namely focal adhesion kinase (FAK) and Src. Here, we show that FAK and Src differ considerably from ACK2 in their abilities to phosphorylate SH3PX1. For example, FAK and Src are more effective kinases for SH3PX1, compared to ACK2. In addition, we show that FAK and Src are able to phosphorylate several carboxyl-terminal truncation mutants of SH3PX1 that are defective for ACK2-catalyzed phosphorylation, suggesting that FAK and Src bind to and phosphorylate different sites on SH3PX1. This was further confirmed by mass spectrometry analysis which identified residues Y177, Y239, Y269, Y294, and Y561, as Src-catalyzed phosphorylation sites, with Y239 as the major site. Given the observed differences exhibited by FAK and Src, versus ACK2, in binding and phosphorylating SH3PX1, it now seems likely that this sorting nexin may be responsible for translating a complicated array of regulatory inputs into the endocytosis and degradation of membrane receptors

An insight into the phylogenetic history of HOX linked gene families in vertebrates

<p>Abstract</p> <p>Background</p> <p>The human chromosomes 2q, 7, 12q and 17q show extensive intra-genomic homology, containing duplicate, triplicate and quadruplicate paralogous regions centered on the HOX gene clusters. The fact that two or more representatives of different gene families are linked with HOX clusters is taken as evidence that these paralogous gene sets might have arisen from a single chromosomal segment through block or whole chromosome duplication events. This would imply that the constituent genes including the HOX clusters reflect the architecture of a single ancestral block (before vertebrate origin) where all of these genes were linked in a single copy.</p> <p>Results</p> <p>In the present study we have employed the currently available set of protein data for a wide variety of vertebrate and invertebrate genomes to analyze the phylogenetic history of 11 multigene families with three or more of their representatives linked to human HOX clusters. A topology comparison approach revealed four discrete co-duplicated groups: group 1 involves the genes from GLI, HH, INHB, IGFBP (cluster-1), and SLC4A families; group 2 involves ERBB, ZNFN1A, and IGFBP (cluster-2) gene families; group 3 involves the HOX clusters and the SP gene family; group 4 involves the integrin beta chain and myosine light chain families. The distinct genes within each co-duplicated group share the same evolutionary history and are duplicated in concert with each other, while the constituent genes of two different co-duplicated groups may not share their evolutionary history and may not have duplicated simultaneously.</p> <p>Conclusion</p> <p>We conclude that co-duplicated groups may themselves be remnants of ancient small-scale duplications (involving chromosomal segments or gene-clusters) which occurred at different time points during chordate evolution. Whereas the recent combination of genes from distinct co-duplicated groups on different chromosomal regions (human chromosomes 2q, 7, 12q, and 17q) is probably the outcome of subsequent rearrangement of genomic segments, including syntenic groups of genes.</p