Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Tandem chimeric transcription as a means to increase protein diversity in the human genome

The “one-gene, one-protein” rule, coined by Beadle and Tatum, has been fundamental to molecular biology. The rule implies that the genetic complexity of an organism depends essentially on its gene number. The discovery, however, that alternative gene splicing and transcription are widespread phenomena dramatically altered our understanding of the genetic complexity of higher eukaryotic organisms; in these, a limited number of genes may potentially encode a much larger number of proteins. Here we investigate yet another phenomenon that may contribute to generate additional protein diversity. Indeed, by relying on both computational and experimental analysis, we estimate that at least 4%–5% of the tandem gene pairs in the human genome can be eventually transcribed into a single RNA sequence encoding a putative chimeric protein. While the functional significance of most of these chimeric transcripts remains to be determined, we provide strong evidence that this phenomenon does not correspond to mere technical artifacts and that it is a common mechanism with the potential of generating hundreds of additional proteins in the human genome.

Resistance to nucleoside analog reverse transcriptase inhibitors mediated by human immunodeficiency virus type 1 p6 protein.

Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the pol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6(gag)-p6(pol) region of HIV-1, placed immediately upstream of pol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6(Gag)), or by changes in activation of the viral protease (p6(Pol)). Duplication of the proline-rich p6(Gag) PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naïve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6(Pol)), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

Lentiviral delivery of the human wild-type tau protein mediates a slow and progressive neurodegenerative tau pathology in the rat brain.

Most models for tauopathy use a mutated form of the Tau gene, MAPT, that is found in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) and that leads to rapid neurofibrillary degeneration (NFD). Use of a wild-type (WT) form of human Tau protein to model the aggregation and associated neurodegenerative processes of Tau in the mouse brain has thus far been unsuccessful. In the present study, we generated an original "sporadic tauopathy-like" model in the rat hippocampus, encoding six Tau isoforms as found in humans, using lentiviral vectors (LVs) for the delivery of a human WT Tau. The overexpression of human WT Tau in pyramidal neurons resulted in NFD, the morphological characteristics and kinetics of which reflected the slow and sporadic neurodegenerative processes observed in sporadic tauopathies, unlike the rapid neurodegenerative processes leading to cell death and ghost tangles triggered by the FTDP-17 mutant Tau P301L. This new model highlights differences in the molecular and cellular mechanisms underlying the pathological processes induced by WT and mutant Tau and suggests that preference should be given to animal models using WT Tau in the quest to understand sporadic tauopathies.

Reactivation of protein aggregates by mortalin and Tid1-the human mitochondrial Hsp70 chaperone system.

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.

Analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in Alzheimer's disease.

Glial fibrillary acidic protein, GFAP, is a major intermediate filament protein of glial cells and major cytoskeletal structure in astrocytes. The entorhinal cortex has a key role in memory function and is one of the first brain areas to reveal hallmark structures of Alzheimer's disease and therefore provides an ideal tissue to investigate incipient neurodegenerative changes. Here we have analyzed age- and disease-related occurrence and composition of GFAP in the human entorhinal cortex by using one- and two-dimensional electrophoresis, Western blots and immunocytochemistry combined with confocal microscopy. A novel monoclonal antibody, GF-02, was characterized that mainly reacted with intact GFAP molecules and indicated that more acidic and soluble GFAP forms were also more susceptible to degradation. GFAP and vimentin increased with aging and in Alzheimer's disease (AD). Two-dimensional electrophoresis and Western blots revealed a complex GFAP pattern, both in aging and AD with different modification and degradation forms. Immunohistochemistry indicated that reactive astrocytes mainly accumulated in relation to neurofibrillary tangles and senile plaques in deeper entorhinal cortex layers. GFAP may be used as an additional but not exclusive diagnostic tool in the evaluation of neurodegenerative diseases because its levels change with age and respond to senile plaque and tangle formation.

Binding of double-strand breaks in DNA by human Rad52 protein.

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

Two-step chromatographic purification of human plasma alpha(1)-acid glycoprotein: its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent.

Alpha1-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (FI*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A*AD, S/A*X0 and F1/A*C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X0 and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells.

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10 % of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

Human epidermal Langerhans cells express the tight junction protein claudin-1 and are present in human genetic claudin-1 deficiency (NISCH syndrome).

Claudin-1 (CLDN1) is a structural tight junction (TJ) protein and is expressed in differentiating keratinocytes and Langerhans cells in the epidermis. Our objective was to identify immunoreactive CLDN1 in human epidermal Langerhans cells and to examine the pattern of epidermal Langerhans cells in genetic human CLDN1 deficiency [neonatal ichthyosis, sclerosing cholangitis (NISCH) syndrome]. Epidermal cells from healthy human skin labelled with CLDN1-specific antibodies were analysed by confocal laser immunofluorescence microscopy and flow cytometry. Skin biopsy sections of two patients with NISCH syndrome were stained with an antibody to CD1a expressed on epidermal Langerhans cells. Epidermal Langerhans cells and a subpopulation of keratinocytes from healthy skin were positive for CLDN1. The gross number and distribution of epidermal Langerhans cells of two patients with molecularly confirmed NISCH syndrome, however, was not grossly altered. Therefore, CLDN1 is unlikely to play a critical role in migration of Langerhans cells (or their precursors) to the epidermis or their positioning within the epidermis. Our findings do not exclude a role of this TJ molecule once Langerhans cells have left the epidermis for draining lymph nodes.

The DNA repair protein ALKBH2 mediates temozolomide resistance in human glioblastoma cells.

INTRODUCTION: Glioblastoma multiforme (GBM; World Health Organization astrocytoma grade IV) is the most frequent and most malignant primary brain tumor in adults. Despite multimodal therapy, all such tumors practically recur during the course of therapy, causing a median survival of only 14.6 months in patients with newly diagnosed GBM. The present study was aimed at examining the expression of the DNA repair protein AlkB homolog 2 (ALKBH2) in human GBM and determining whether it could promote resistance to temozolomide chemotherapy. METHODS: ALKBH2 expression in GBM cell lines and in human GBM was determined by quantitative real-time PCR (qRT-PCR) and gene expression analysis, respectively. Drug sensitivity was assessed in GBM cells overexpressing ALKBH2 and in cells in which ALKBH2 expression was silenced by small-interfering (si)RNA. ALKBH2 expression following activation of the p53 pathway was examined by western blotting and qRT-PCR. RESULTS: ALKBH2 was abundantly expressed in established GBM cell lines and human GBM, and temozolomide exposure increased cellular ALKBH2 expression levels. Overexpression of ALKBH2 in the U87 and U251 GBM cell lines enhanced resistance to the methylating agents temozolomide and methyl methanesulfonate but not to the nonmethylating agent doxorubicin. Conversely, siRNA-mediated knockdown of ALKBH2 increased sensitivity of GBM cells to temozolomide and methyl methanesulfonate but not to doxorubicin or cisplatin. Nongenotoxic activation of the p53 pathway by the selective murine double minute 2 antagonist nutlin-3 caused a significant decrease in cellular ALKBH2 transcription levels. CONCLUSION: Our findings identify ALKBH2 as a novel mediator of temozolomide resistance in human GBM cells. Furthermore, we place ALKBH2 into a new cellular context by showing its regulation by the p53 pathway.

Conformational changes modulate the activity of human RAD51 protein.

Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homologue, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity.

Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression.

Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.