'Drc', a structurally novel ssDNA-binding transcription regulator of N4-related bacterial viruses.

Bacterial viruses encode a vast number of ORFan genes that lack similarity to any other known proteins. Here, we present a 2.20 Å crystal structure of N4-related Pseudomonas virus LUZ7 ORFan gp14, and elucidate its function. We demonstrate that gp14, termed here as Drc (ssDNA-binding RNA Polymerase Cofactor), preferentially binds single-stranded DNA, yet contains a structural fold distinct from other ssDNA-binding proteins (SSBs). By comparison with other SSB folds and creation of truncation and amino acid substitution mutants, we provide the first evidence for the binding mechanism of this unique fold. From a biological perspective, Drc interacts with the phage-encoded RNA Polymerase complex (RNAPII), implying a functional role as an SSB required for the transition from early to middle gene transcription during phage infection. Similar to the coliphage N4 gp2 protein, Drc likely binds locally unwound middle promoters and recruits the phage RNA polymerase. However, unlike gp2, Drc does not seem to need an additional cofactor for promoter melting. A comparison among N4-related phage genera highlights the evolutionary diversity of SSB proteins in an otherwise conserved transcription regulation mechanism.

Structures of the heart specific SERCA2a Ca2+-ATPase.

The sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA-stabilized E2-AlF4- form (3.3 Å) and the Ca2+-occluded [Ca2]E1-AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform-specific residues are acceptor sites for post-translational modifications. In addition, molecular dynamics simulations predict that isoform-specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform-specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.

Rational design of small molecules that modulate the transcriptional function of the response regulator PhoP.

The response regulator PhoP, which is part of the PhoP/PhoQ two-component system, regulates the expression of multiple genes involved in controlling virulence in Salmonella enterica serovar Typhimurium and other species of Gram-negative bacteria. Modulating the phosphorylation-mediated dimerization in the receiver domain may interfere with the transcriptional function of PhoP. In this study, we analyzed the therapeutic potential of the PhoP receiver domain by exploring it as a potential target for drug design. The structural information was then applied to identify the first hit compounds from commercial chemical libraries by combining pharmacophore modelling and docking methods with a GFP (Green Fluorescent Protein)-based promoter-fusion bioassay. In total, one hundred and forty compounds were selected, purchased, and tested for biological activity. Several novel scaffolds showed acceptable potency to modulate the transcriptional function of PhoP, either by enhancing or inhibiting the expression of PhoP-dependent genes. These compounds may be used as the starting point for developing modulators that target the protein-protein interface of the PhoP protein as an alternative strategy against antibiotic resistance.

Computational Studies of the Active and Inactive Regulatory Domains of Response Regulator PhoP Using Molecular Dynamics Simulations.

The response regulator PhoP is part of the PhoP/PhoQ two-component system, which is responsible for regulating the expression of multiple genes involved in controlling virulence, biofilm formation, and resistance to antimicrobial peptides. Therefore, modulating the transcriptional function of the PhoP protein is a promising strategy for developing new antimicrobial agents. There is evidence suggesting that phosphorylation-mediated dimerization in the regulatory domain of PhoP is essential for its transcriptional function. Disruption or stabilization of protein-protein interactions at the dimerization interface may inhibit or enhance the expression of PhoP-dependent genes. In this study, we performed molecular dynamics simulations on the active and inactive dimers and monomers of the PhoP regulatory domains, followed by pocket-detecting screenings and a quantitative hot-spot analysis in order to assess the druggability of the protein. Consistent with prior hypothesis, the calculation of the binding free energy shows that phosphorylation enhances dimerization of PhoP. Furthermore, we have identified two different putative binding sites at the dimerization active site (the α4-ß5-α5 face) with energetic "hot-spot" areas, which could be used to search for modulators of protein-protein interactions. This study delivers insight into the dynamics and druggability of the dimerization interface of the PhoP regulatory domain, and may serve as a basis for the rational identification of new antimicrobial drugs.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.

Viral interference of the bacterial RNA metabolism machinery.

In a recent publication, we reported a unique interaction between a protein encoded by the giant myovirus phiKZ and the Pseudomonas aeruginosa RNA degradosome. Crystallography, site-directed mutagenesis and interactomics approaches revealed this 'degradosome interacting protein' or Dip, to adopt an 'open-claw' dimeric structure that presents acidic patches on its outer surface which hijack 2 conserved RNA binding sites on the scaffold domain of the RNase E component of the RNA degradosome. This interaction prevents substrate RNAs from being bound and degraded by the RNA degradosome during the virus infection cycle. In this commentary, we provide a perspective into the biological role of Dip, its structural analysis and its mysterious evolutionary origin, and we suggest some therapeutic and biotechnological applications of this distinctive viral protein.

Five lung nodules, four different pathologic findings: not all lung lesions are the same.

A patient with a history of colon adenocarcinoma was referred for resection of two lung lesions suspect for metastases. Three additional nodular areas were removed by wedge excision. Pathologic examination revealed four different diagnoses. This particular case demonstrates that the number of lesions may be underestimated on preoperative imaging, even on high-resolution computed tomography and positron emission tomography. Equally, pathologic examination of all detected lesions is vitally important to decide optimal treatment and to determine prognosis. This should be taken into account when discussing alternative treatment options to surgery such as stereotactic radiotherapy and radiofrequency ablation.

Structural elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA degradosome.

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 Šcrystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells.

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.

Validation of APRI and FIB-4 score in an Antwerp cohort of chronic hepatitis C patients.

BACKGROUND AND AIMS: Evaluation of liver fibrosis in chronic hepatitis C patients guides clinical decision-making. The aim of this study is to validate APRI and FIB-4, two easily calculated noninvasive tests to predict fibrosis, in chronic HCV patients using biopsy as a gold standard and to compare accuracy between HCV monoinfected and HIV/HCV coinfected patients. PATIENTS AND METHODS: We retrospectively studied HCV patients of two centres who underwent liver biopsy. Liver fibrosis was staged according to METAVIR. RESULTS: 136 patients were included. The AUROC of FIB-4 (0.896) to discriminate F0-F2 vs. F3-F4 was significantly higher (p=0.0186) than the AUROC of APRI (0.842). The difference in AUROC between HIV-negative and positive patients was not significant for APRI (p=0.471), nor for FIB-4 (p=0.495). Performance status was lower in HIV-positive patients with 46.7% and 69.0% of patients correctly classified using APRI and FIB-4, compared to 56.6% and 73.6% in HIV-negative patients, respectively. Conversion of transaminase values from one hospital to the other did not significantly change the AUROC of FIB-4 (p=0.928). CONCLUSIONS: APRI and FIB-4 have a better performance status in HCV monoinfected patients compared to HIV/HCV coinfected patients. FIB-4 has a better AUROC compared to APRI and is the preferred noninvasive fibrosis score to discriminate between F0-F2 and F3-F4. Different hospitals should use their local absolute serum transaminase values without conversion.

Mechanism of Nonpolar Model Substances to Inhibit Primary Gushing Induced by Hydrophobin HFBI.

In this work, the interactions of a well-studied hydrophobin with different types of nonpolar model substances and their impact on primary gushing is evaluated. The nature, length, and degree of saturation of nonpolar molecules are key parameters defining the gushing ability or inhibition. When mixed with hydrophobins, the nonpolar molecule-hydrophobin assembly acts as a less gushing or no gushing system. This effect can be explained in the framework of a competition effect between non-polar systems and CO2 to interact with the hydrophobic patch of the hydrophobin. Interactions of these molecules with hydrophobins are promoted as a result of the similar size of the nonpolar molecules with the hydrophobic patch of the protein, at the expense of the formation of nanobubbles with CO2. In order to prove the presence of interactions and to unravel the mechanisms behind them, a complete set of experimental techniques was used. Surface sensitive techniques clearly show the presence of the interactions, whose nature is not covalent nor hydrogen bonding according to infrared spectroscopy results. Interactions were also reflected by particle size analysis in which mixtures of particles displayed larger size than their pure component counterparts. Upon mixing with nonpolar molecules, the gushing ability of the protein is significantly disrupted.

HIV-1 integrase variants retarget viral integration and are associated with disease progression in a chronic infection cohort.

Distinct integration patterns of different retroviruses, including HIV-1, have puzzled virologists for over 20 years. A tetramer of the viral integrase (IN) assembles on the two viral cDNA ends, docks onto the target DNA (tDNA), and catalyzes viral genome insertion into the host chromatin. We identified the amino acids in HIV-1 IN that directly contact tDNA bases and affect local integration site sequence selection. These residues also determine the propensity of the virus to integrate into flexible tDNA sequences. Remarkably, natural polymorphisms INS119G and INR231G retarget viral integration away from gene-dense regions. Precisely these variants were associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution, but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors.

In silico, in vitro and cellular analysis with a kinome-wide inhibitor panel correlates cellular LRRK2 dephosphorylation to inhibitor activity on LRRK2.

Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase inhibitors.

LEDGINs, non-catalytic site inhibitors of HIV-1 integrase: a patent review (2006 - 2014).

INTRODUCTION: Integration of the viral genome into the host cell chromatin is a central step in the replication cycle of the HIV. Blocking the viral integrase (IN) enzyme therefore provides an attractive therapeutic strategy, as evidenced by the recent clinical approval of three IN strand transfer inhibitors. Viral resistance and cross-resistance among these inhibitors, however, warrant the search for compounds targeting HIV integration through alternative mechanisms of action. AREAS COVERED: The most potent class of allosteric IN inhibitors was independently identified at the University of Leuven, Belgium, and at Boehringer Ingelheim, Canada. These compounds, coined LEDGINs (after the lens epithelium-derived growth factor/p75 cofactor binding pocket on IN) or non-catalytic site IN inhibitors (NCINIs) by the respective groups, have shown remarkable antiviral activity. This review provides a brief introduction to the compound class and discusses the recent patent literature (2006 to the present). EXPERT OPINION: LEDGINs are still early in development. Trials with clinical candidate BI-224436 were put on hold despite promising results. Literature, however, reveals that almost all major pharmaceutical companies active in the treatment of HIV/AIDS have taken a significant interest in this class. As a result, several of these inhibitors may soon enter clinical trials.

Identification of small peptides inhibiting the integrase-LEDGF/p75 interaction through targeting the cellular co-factor.

The integration of the viral DNA into the host genome is one of the essential steps in the HIV replication cycle. This process is mediated by the viral enzyme integrase (IN) and lens epithelium-derived growth factor (LEDGF/p75). LEDGF/p75 has been identified as a crucial cellular co-factor of integration that acts by tethering IN to the cellular chromatin. Recently, circular peptides were identified that bind to the C-terminal domain of IN and disrupt the interaction with LEDGF/p75. Starting from the circular peptides, we identified a short peptidic sequence able to inhibit the LEDGF/p75-IN interaction at low µM concentration through its binding to the IN binding site of LEDGF/p75. This discovery can lead to the synthesis of peptidomimetics with high anti-HIV activity targeting the cellular co-factor LEDGF/p75 and not the viral protein IN.

Tuning the size and properties of ClyA nanopores assisted by directed evolution.

Nanopores have recently emerged as powerful tools in single-molecule investigations. Biological nanopores, however, have drawbacks, including a fixed size and limited stability in lipid bilayers. Inspired by the great success of directed evolution approaches in tailoring enzyme properties, in this work we evolved Cytolysin A from Salmonella typhi (ClyA) to a high level of soluble expression and desired electrical properties in lipid bilayers. Evolved ClyA nanopores remained open up to -150 mV applied potential, which allowed the detailed characterization of folded proteins by ionic current recordings. Remarkably, we also found that ClyA forms several nanopore species; among which we could isolate and characterize three nanopore types most likely corresponding to the 12mer, 13mer, and 14mer oligomeric forms of ClyA. Protein current blockades to the three ClyA nanopores showed that subnanometer variations in the diameter of nanopores greatly affect the recognition of analyte proteins.

Mapping the binding interface between an HIV-1 inhibiting intrabody and the viral protein Rev.

HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb190) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb190-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb190 and the Rev multimerization domains were evaluated in different assays measuring Nb190-Rev interaction or viral production. Seven residues within Nb190 and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb190-Rev structural interface. This Nb190-Rev interaction model can guide further studies of the Nb190 effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb190 epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.

Understanding the role of defective invertases in plants: tobacco Nin88 fails to degrade sucrose.

Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs.

Rapid Conformational Fluctuations of Disordered HIV-1 Fusion Peptide in Solution.

The conformationally flexible fusion peptide (FP) of HIV-1 is indispensible for viral infection of host cells, due to its ability to insert into and tightly couple with phospholipid membranes. There are conflicting reports on the membrane-associated structure of FP, and solution structure information is limited, yet such a structure is the target for a novel class of antiretroviral inhibitors. An ensemble of explicit solvent molecular dynamics simulations, initiated from a disordered HIV-1 FP (aggregate time of ∼30 µs), revealed that while the vast majority of conformations predominantly lack secondary structure, both spontaneous formation and rapid interconversion of local secondary structure elements occur, highlighting the structural plasticity of the peptide. Therefore, even at this rapid time scale, FP constitutes a diverse and flexible conformational ensemble in solution. Secondary structure clustering reveals that the most prominent ordered elements are α- and 3-10-helical subsets of membrane-bound conformations, while trace populations within 2 Å RMSD of all complete membrane-bound conformations are found to pre-exist in the solution ensemble. Since inhibitor bound conformations of FP are only rarely found, FP inhibitors could function by modulating the conformational ensemble and binding to nonfusogenic FP structures. A thermodynamic characterization of the most prominent ordered nonfusogenic structures could facilitate the future design of improved FP inhibitors.

An integrated fragment based screening approach for the discovery of small molecule modulators of the VWF-GPIbα interaction.

An integrated approach comprising STD NMR screening, pharmacophore based analogue selection and a bioassay is presented for the discovery of a stabilizer of the clinically relevant VWF-GPIbα protein-protein interaction.