A workflow for in silico design of hIL-10 and ebvIL-10 inhibitors using well-known miniprotein scaffolds.

The over-expression of immune-suppressors such as IL-10 is a crucial landmark in both tumor progression, and latent viral and parasite infection. IL-10 is a multifunctional protein. Besides its immune-cell suppressive function, it also promotes B-cell tumorigenesis of lymphomas and melanoma. Human pathogens like unicellular parasites and viruses that remain latent inside B cells promote the over-expression of hIL-10 upon infection, which inhibits cell-mediated immune surveillance, and at the same time mediates B cell proliferation. The B-cell specific oncogenic latent virus Epstein-Barr virus (EBV) encodes a viral homologue of hIL-10 (ebvIL-10), expressed during lytic viral proliferation. Once expressed, ebvIL-10 inhibits cell-mediated immune surveillance, assuring EBV re-infection. During long-term latency, EBV-infected B cells over-express hIL-10 to assure B-cell proliferation, occasionally inducing EBV-mediated lymphomas. The amino acid sequences of hIL-10 and ebvIL-10 are more than 80% identical and thus have a very similar tridimensional structure. Based on their published crystallographic structures bound to their human receptor IL10R1, we report a structure-based design of hIL-10 and ebvIL-10 inhibitors based on 3 loops from IL10R1 that establish specific hydrogen bonds with the two IL10s. We have grafted these loops onto a permissible loop in three well-known miniprotein scaffolds-the Conus snail toxin MVIIA, the plant-derived trypsin inhibitor EETI, and the human appetite modulator AgRP. Our computational workflow described in detail below was invigorated by the negative and positive controls implemented, and therefore paves the way for future in vitro and in vivo validation assays of the IL-10 inhibitors engineered.

Consideration of Epstein-Barr Virus-Encoded Noncoding RNAs EBER1 and EBER2 as a Functional Backup of Viral Oncoprotein Latent Membrane Protein 1.

The Epstein-Barr virus (EBV)-encoded noncoding RNAs EBER1 and EBER2 are highly abundant through all four latency stages of EBV infection (III-II-I-0) and have been associated with an oncogenic phenotype when expressed in cell lines cultured in vitro. In vivo, EBV-infected B cells derived from freshly isolated lymphocytes show that EBER1/2 deletion does not impair viral latency. Based on published quantitative proteomics data from BJAB cells expressing EBER1 and EBER2, we propose that the EBERs, through their activation of AKT in a B-cell-specific manner, are a functionally redundant backup of latent membrane protein 1 (LMP1)-an essential oncoprotein in EBV-associated malignancies, with a main role in AKT activation. Our proposed model may explain the lack of effect on viral latency establishment in EBER-minus EBV infection.

Two microtubule-plus-end binding proteins LIS1-1 and LIS1-2, homologues of human LIS1 in Neurospora crassa.

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1.

Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2.

In Epstein-Barr virus (EBV) latent infection, the EBV-encoded RNAs EBER1 and EBER2 accumulate in the host cell nucleus to ~10(6) copies. While the expression of EBERs in cell lines is associated with transformation, a mechanistic explanation of their roles in EBV latency remains elusive. To identify EBER-specific gene expression features, we compared the proteome and mRNA transcriptome from BJAB cells (an EBV-negative B lymphoma cell line) stably transfected with an empty plasmid or with one carrying both EBER genes. We identified ~1800 proteins with at least 2 SILAC pair measurements, of which only 8 and 12 were up- and downregulated ≥ 2-fold, respectively. One upregulated protein was PIK3AP1, a B-cell specific protein adapter known to activate the PI3K-AKT signaling pathway, which regulates alternative splicing and translation in addition to its pro-survival effects. In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change. We instead observed isoform switch events. We validated the most relevant findings with biochemical assays. These corroborated the upregulation of PIK3AP1 and AKT activation in BJAB cells expressing high levels of both EBERs and EBNA1 (a surrogate of Burkitt's lymphoma EBV latency I) relative to those expressing only EBNA1. The mRNA-seq data in these cells showed multiple upregulated oncogenes whose mRNAs are enriched for 3´-UTR AU-rich elements (AREs), such as ccl3, ccr7, il10, vegfa and zeb1. The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas. In EBV latency, ZEB1 represses the transcription of ZEBRA, an EBV lytic phase activation factor. We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs. Thus, the ~10(6) copies of EBER1 may promote not only cell proliferation due to an increase in the levels of ARE-containing genes like ccl3, ccr7, il10, and vegfa, but also the maintenance of latency, through higher levels of zeb1.

RNA families in Epstein-Barr virus.

Epstein-Barr virus (EBV) is a tumorigenic human γ-herpesvirus, which produces several known structured RNAs with functional importance: two are implicated in latency maintenance and tumorigenic phenotypes, EBER1 and EBER2; a viral small nucleolar RNA (v-snoRNA1) that may generate a small regulatory RNA; and an internal ribosomal entry site in the EBNA1 mRNA. A recent bioinformatics and RNA-Seq study of EBV identified two novel EBV non-coding (nc)RNAs with evolutionary conservation in lymphocryptoviruses and likely functional importance. Both RNAs are transcribed from a repetitive region of the EBV genome (the W repeats) during a highly oncogenic type of viral latency. One novel ncRNA can form a massive (586 nt) hairpin, while the other RNA is generated from a short (81 nt) intron and is found in high abundance in EBV-infected cells.

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus.

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

AUF1/hnRNP D is a novel protein partner of the EBER1 noncoding RNA of Epstein-Barr virus.

Epstein-Barr virus (EBV)-infected cells express two noncoding RNAs called EBV-encoded RNA (EBER) 1 and EBER2. Despite their high abundance in the nucleus (about 10(6) copies), the molecular function of these noncoding RNAs has remained elusive. Here, we report that the insertion into EBER1 of an RNA aptamer that binds the bacteriophage MS2 coat protein allows the isolation of EBER1 and associated protein partners. By combining MS2-mediated selection with stable isotope labeling of amino acids in cell culture (SILAC) and analysis by mass spectrometry, we identified AUF1 (AU-rich element binding factor 1)/hnRNP D (heterogeneous nuclear ribonucleoprotein D) as an interacting protein of EBER1. AUF1 exists as four isoforms generated by alternative splicing and is best known for its role in destabilizing mRNAs upon binding to AU-rich elements (AREs) in their 3' untranslated region (UTR). Using UV crosslinking, we demonstrate that predominantly the p40 isoform of AUF1 interacts with EBER1 in vivo. Electrophoretic mobility shift assays show that EBER1 can compete for the binding of the AUF1 p40 isoform to ARE-containing RNA. Given the high abundance of EBER1 in EBV-positive cells, EBER1 may disturb the normal homeostasis between AUF1 and ARE-containing mRNAs or compete with other AUF1-interacting targets in cells latently infected by EBV.

A modular approach to the design of protein-based smart gels.

The modular nature of repeat proteins makes them a versatile platform for the design of smart materials with predetermined properties. Here, we present a general strategy for combining protein modules with specified stability and function into arrays for the assembly of stimuli-responsive gels. We have designed tetratricopeptide repeat (TPR) arrays which contain peptide-binding modules that specify the strength and reversibility of network crosslinking in combination with spacer modules that specify crosslinking geometry and overall stability of the array. By combining such arrays with multivalent peptide ligands, self-supporting stimuli-responsive gels are formed. Using microrheology, we characterized the kinetics of gelation as a function of concentration and stoichiometry of the components. We also show that such gels are effective in encapsulating and releasing small molecules. Moreover, TPR gels alone are fully compatible with cell growth, whereas gels loaded with an anticancer compound release the compound, resulting in cell death. Thus, we have demonstrated that this new class of tunable biomaterials is ripe for further development as tissue engineering and drug delivery platform.

A compound that inhibits the HOP-Hsp90 complex formation and has unique killing effects in breast cancer cell lines.

The chaperone Hsp90 is required for the correct folding and maturation of certain "client proteins" within all cells. Hsp90-mediated folding is particularly important in cancer cells, because upregulated or mutant oncogenic proteins are often Hsp90 clients. Hsp90 inhibitors thus represent a route to anticancer agents that have the potential to be active against several different types of cancer. Currently, various Hsp90 inhibitors that bind to Hsp90 at its ATP-binding site are in preclinical and clinical trials. Some of the most promising Hsp90 ATP-binding site inhibitors are the well characterized geldanamycin derivative 17-AAG and the recently described compounds PU-H71 and NVP-AUY922. An undesirable characteristic of these compounds is the transcriptional upregulation of Hsp70 that has prosurvival effects. Here we characterize the activity of a new type of chaperone inhibitor, 1,6-dimethyl-3-propylpyrimido[5,4-e][1,2,4]triazine-5,7-dione (named C9 for simplicity). Using purified protein components in vitro, C9 prevents Hsp90 from interacting with the cochaperone HOP and is thus expected to impair the Hsp90-dependent folding pathway in vivo. We show that this compound is effective in killing various breast cancer cell lines including the highly metastatic MDA-MB-231. An important property of this compound is that it does not induce the transcriptional upregulation of Hsp70. Moreover, when cells are treated with a combination of C9 and either 17-AAG or NVP-AUY922, the overexpression of Hsp70 is counteracted considerably and C9's lethal-IC50 decreases compared to its value when added alone.

Proteomic characterization of Her2/neu-overexpressing breast cancer cells.

The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.

Autophosphorylation properties of inactive and active JNK2.

The c-Jun N-terminal kinases (JNKs) are ubiquitous proteins that phosphorylate their substrates, such as transcription factors, in response to physical stress, cytokines or UV radiation. This leads to changes in gene expression, ensuing either cell cycle progression or apoptosis. Active phospho JNK1 is the main in vivo kinase component of the JNK cascade, whereas JNK2 is presumed not to participate as a kinase during JNK signalling. However, there is evidence that JNK isoforms interact functionally in vivo. Also, a recent chemical genetics investigation has confirmed that JNK transient activation leads to cellular proliferation, whereas a sustained one is pro-apoptotic. Here we investigate the phosphorylation pattern of JNK2, with protein biochemistry tools and tandem mass spectrometry. We choose to focus on JNK2 because of its reported constitutive activity in glioma cells. Our results indicate that purified JNK2 from transfected nonstressed 293T cells is a mixture of the mono-sites pThr183 and pTyr185 of its activation loop and of pThr386 along its unique C-terminal region. Upon UV stimulation, its phosphorylation stoichiometry is upregulated on the activation loop, generating a mixture of mono-pTyr185 and the expected dual-pThr183/pTyr185 species, with the pThr386 specie present but unaltered respect to the basal conditions.

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.