Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins.

Open reading frames in the Plasmodium falciparum genome encode domains homologous to the adhesive domains of the P. falciparum EBA-175 erythrocyte-binding protein (eba-175 gene product) and those of the Plasmodium vivax and Plasmodium knowlesi Duffy antigen-binding proteins. These domains are referred to as Duffy binding-like (DBL), after the receptor that determines P. vivax invasion of Duffy blood group-positive human erythrocytes. Using oligonucleotide primers derived from short regions of conserved sequence, we have developed a reverse transcription-PCR method that amplifies sequences encoding the DBL domains of expressed genes. Products of these reverse transcription-PCR amplifications include sequences of single-copy genes (including eba-175) and variably transcribed genes that cross-hybridize to multiple regions of the genome. Restriction patterns of the multicopy genes show a high degree of polymorphism among different parasite lines, whereas single-copy genes are generally conserved. Characterization of the single-copy genes has identified a gene (ebl-1) that is related to eba-175 and is likely to be involved in erythrocyte invasion.

A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei.

We have identified a class of proteins that bind single-stranded telomeric DNA and are required for the nuclear organization of telomeres and/or telomere-associated proteins. Rlf6p was identified by its sequence similarity to Gbp1p, a single-stranded telomeric DNA-binding protein from Chlamydomonas reinhardtii. Rlf6p and Gbp1p bind yeast single-stranded G-strand telomeric DNA. Both proteins include at least two RNA recognition motifs, which are found in many proteins that interact with single-stranded nucleic acids. Disruption of RLF6 alters the distribution of repressor/activator protein 1 (Rap1p), a telomere-associated protein. In wild-type yeast cells, Rap1p localizes to a small number of perinuclear spots, while in rlf6 cells Rap1p appears diffuse and nuclear. Interestingly, telomere position effect and telomere length control, which require RAP1, are unaffected by rlf6 mutations, demonstrating that Rap1p localization can be uncoupled from other Rap1p-dependent telomere functions. In addition, expression of Chlamydomonas GBP1 restores perinuclear, punctate Rap1p localization in rlf6 mutant cells. The functional complementation of a fungal gene by an algal gene suggests that Rlf6p and Gbp1p are members of a conserved class of single-stranded telomeric DNA-binding proteins that influence nuclear organization. Furthermore, it demonstrates that, despite their unusual codon bias, C. reinhardtii genes can be efficiently translated in Saccharomyces cerevisiae cells.

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression.

The transient expression of the retinoblastoma protein (Rb) regulates the transcription of a variety of growth-control genes, including c-fos, c-myc, and the gene for transforming growth factor beta 1 via discrete promoter sequences termed retinoblastoma control elements (RCE). Previous analyses have shown that Sp1 is one of three RCE-binding proteins identified in nuclear extracts and that Rb functionally interacts with Sp1 in vivo, resulting in the "superactivation" of Sp1-mediated transcription. By immunochemical and biochemical criteria, we report that an Sp1-related transcription factor, Sp3, is a second RCE-binding protein. Furthermore, in transient cotransfection assays, we report that Rb "superactivates" Sp3-mediated RCE-dependent transcription in vivo and that levels of superactivation are dependent on the trans-activator (Sp1 or Sp3) studied. Using expression vectors carrying mutated Rb cDNAs, we have identified two portions of Rb required for superactivation: (i) a portion of the Rb "pocket" (amino acids 614-839) previously determined to be required for physical interactions between Rb and transcription factors such as E2F-1 and (ii) a novel amino-terminal region (amino acids 140-202). Since both of these regions of Rb are targets of mutation in human tumors, our data suggest that superactivation of Sp1/Sp3 may play a role in Rb-mediated growth suppression and/or the induction of differentiation.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-IR rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK 1/2 phosphorylation. Using toll-like receptor (th-)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R. (C) 2005 Elsevier GmbH. All rights reserved.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.

Attenuation of skeletal muscle atrophy in cancer cachexia by d-myo-inositol 1,2,6-triphosphate

PURPOSE: To determine the effectiveness of the polyanionic, metal binding agent D-myo-inositol-1,2,6-triphosphate (alpha trinositol, AT), and its hexanoyl ester (HAT), in tissue wasting in cancer cachexia. METHODS: The anti-cachexic effect was evaluated in the MAC16 tumour model. RESULTS: Both AT and HAT attenuated the loss of body weight through an increase in the nonfat carcass mass due to an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. The decrease in protein degradation was associated with a decrease in activity of the ubiquitin-proteasome proteolytic pathway and caspase-3 and -8. Protein synthesis was increased due to attenuation of the elevated autophosphorylation of double-stranded RNA-dependent protein kinase, and of eukaryotic initiation factor 2alpha together with hyperphosphorylation of eIF4E-binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2. In vitro, AT completely attenuated the protein degradation in murine myotubes induced by both proteolysis-inducing factor and angiotensin II. CONCLUSION: These results show that AT is a novel therapeutic agent with the potential to alleviate muscle wasting in cancer patients.

EnDNA-Prot:identification of DNA-binding proteins by applying ensemble learning

DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97-9.52% in ACC and 0.08-0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83-16.63% in terms of ACC and 0.02-0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public. © 2014 Ruifeng Xu et al.

Identifying DNA-binding proteins by combining support vector machine and PSSM distance transformation

Background: DNA-binding proteins play a pivotal role in various intra- and extra-cellular activities ranging from DNA replication to gene expression control. Identification of DNA-binding proteins is one of the major challenges in the field of genome annotation. There have been several computational methods proposed in the literature to deal with the DNA-binding protein identification. However, most of them can't provide an invaluable knowledge base for our understanding of DNA-protein interactions. Results: We firstly presented a new protein sequence encoding method called PSSM Distance Transformation, and then constructed a DNA-binding protein identification method (SVM-PSSM-DT) by combining PSSM Distance Transformation with support vector machine (SVM). First, the PSSM profiles are generated by using the PSI-BLAST program to search the non-redundant (NR) database. Next, the PSSM profiles are transformed into uniform numeric representations appropriately by distance transformation scheme. Lastly, the resulting uniform numeric representations are inputted into a SVM classifier for prediction. Thus whether a sequence can bind to DNA or not can be determined. In benchmark test on 525 DNA-binding and 550 non DNA-binding proteins using jackknife validation, the present model achieved an ACC of 79.96%, MCC of 0.622 and AUC of 86.50%. This performance is considerably better than most of the existing state-of-the-art predictive methods. When tested on a recently constructed independent dataset PDB186, SVM-PSSM-DT also achieved the best performance with ACC of 80.00%, MCC of 0.647 and AUC of 87.40%, and outperformed some existing state-of-the-art methods. Conclusions: The experiment results demonstrate that PSSM Distance Transformation is an available protein sequence encoding method and SVM-PSSM-DT is a useful tool for identifying the DNA-binding proteins. A user-friendly web-server of SVM-PSSM-DT was constructed, which is freely accessible to the public at the web-site on http://bioinformatics.hitsz.edu.cn/PSSM-DT/.

Structural and Functional Analysis of the Caspase –dependent and –independent Domains of the X-linked Inhibitor of Apoptosis Protein in Inflammatory Breast Cancer Tumor Biology

Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance.

Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.

Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Dissecting the role of Profilin-1 in microglial cell function: the impact on ROS production

Microglial cells are the resident immune cells of central nervous system (CNS) and the major players in neuroinflammation. These cells are also responsible for surveilling the neuronal microenvironment, and upon injury to the CNS they change their morphology and molecular profile and become activated. Activated status is associated with microglia proliferation, migration to injury foci, increased phagocytic capacity, production and release of reactive oxygen species (ROS), cytokines (pro- or anti-inflammatory) and reactive nitrogen species. Microglia activation is crucial for tissue repair in the healthy brain. However, their chronic activation or deregulation might contribute for the pathophysiology of neurodegenerative diseases. A better understanding of the mechanisms underlying microglial cell activation is important for defining targets and develop appropriate therapeutic strategies to control the chronic activation of microglia. It has been observed an increase in profilin (Pfn) mRNA in microglial cells in the rat hippocampus after unilateral ablation of its major extrinsic input, the entorhinal cortex. This observation suggested that Pfn might be involved in microglia activation. Pfn1 is an actin binding protein that controls assembly and disassembly of actin filaments and is important for several cellular processes, including, motility, cell proliferation and survival. Here, we studied the role of Pfn1 in microglial cell function. For that, we used primary cortical microglial cell cultures and microglial cell lines in which we knocked down Pfn1 expression and assessed the activation status of microglia, based on classical activation markers, such as: phagocytosis, glutamate release, reactive oxygen species (ROS), pro- and anti-inflammatory cytokines. We demonstrated that Pfn1 (i) is more active in hypoxia-challenged microglia, (ii) modulates microglia pro- and anti-inflammatory signatures and (iii) plays a critical role in ROS generation in microglia. Altogether, we conclude that Pfn1 is a key protein for microglia homeostasis, playing an essential role in their activation, regardless the polarization into a pro or anti-inflammatory signature.

C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition