An Allosteric Mechanism for Inhibiting HIV-1 Integrase with a Small Molecule

HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the small-molecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN

HIV-1 Integrase-DNA Recognition Mechanisms

Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review

Oksymetriscreening av nyfødte - Tidligere diagnostikk av medfødt hjertesykdom

Bakgrunn/emne: Pulsoksymetri av barn i løpet av første levedøgn kan hjelpe til å avdekke ikke diagnostiserte medfødte hjertesykdommer. Pulsoksymetri er ikke en rutineundersøkelse ved fødeavdelingen ved Ullevål Universitetssykehus. I dette kvalitetsforbedringsprosjektet ville vi innføre pulsoksymetriscreening av nyfødte, med ønske om å fremskynde diagnostikken av medfødte hjertefeil ved Ullevål sykehus. Kunnskapsgrunnlaget: Det ble i hovedsak studert artikler i form av prospektive studier og systematiske oversikter. Søkemotorene Pubmed/Medline, Cochrane og Embase ble brukt. Ut fra inklusjonskriteriene ble 5 studier og 2 systematiske oversikter inkludert. Det ble også forsøkt å søke etter en nasjonal evidensbasert retningslinje for pulsoksymetri screening av nyfødte uten treff, det bekrefter at det inntil videre ikke eksisterer noen nasjonale retningslinjer når det gjelder pulsoksymetriscreening av nyfødte. Tiltak og metode: Vi valgte å beskrive et kvalitetsforbedringsprosjekt som innebar en endring av bestående retningslinjer, der vi innførte et nytt element; oksymetriscreening i retningslinjene for klinisk undersøkelse av nyfødte. For å sikre at den nye prosedyren følges skal resultatet av målingen føres inn i elektronisk pasientjournal (prosessindikator). Resultatindikatoren vår er ”andelen barn med medfødt hjertesykdom diagnostisert før utskrivelse fra barselavdelingen”. Organisering: Vi organiserte prosjektet etter Demnings kvalitetshjul, PDSA- hjulet (Plan, Do, Study og Act), en syklisk kvalitetsendringsmodell som baserer seg på samarbeid med ledelse og faggrupper, og evaluering og eventuell korrigering av prosjektet. Basert på en algoritme presentert i en artikkel av Meberg et al. laget vi et flytskjema som viste hvordan man skal gå videre med resultatene fra oksymetriscreeningen. Resultat og vurdering: Kunnskapsgrunnlaget støtter bruk av oxymetriscreening av nyfødte. Dette vil føre til tidligere diagnostisering av barn med hjertefeil og gi mulighet for tidligere intervensjon. Dette vil gi en stor gevinst for de som er rammet av medfødt hjertefeil. Prosedyren er ikke invasiv og krever lite økonomiske ressurser og ingen ekstra personell. Det er sannsynelig at den nye retningslinjen vil bli innført ved barselavdelingen ved Ullevål Universitetssykehus

Structural basis for high-affinity binding of LEDGF PWWP to mononucleosomes

Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c

SETD2-dependent histone H3K36 trimethylation is required for homologous recombination repair and genome stability

Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis. © 2014 The Authors

Cryo-electron microscopy of chromatin biology

The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein-DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward. In this review, the authors highlight recent advances in the architectural understanding of chromatin biology elucidated by EM

Lagring av hornhinnevev

The first successful penetrating keratoplasty in a human was performed by Eduard Zirm in 1905. In 1935, V. P. Filatov was the first to report successful storage of whole eyeglobes at 4°C. The opportunity to store corneal tissue is crucial for extensive use of keratoplasty as treatment for corneal blindness. Since the endothelium′s importance for corneal function has been known for the last five decades, most research performed on corneal storage media has focused on the preservation of the donor endothelium. As an intact epithelium also is important for graft survival, further knowledge is needed regarding better preservation of the epithelium. The effect of corneal storage on keratocyte viability is still not certain. At present, hypotherm storage, mainly Optisol-GS, and organ culture are the most frequently used storage techniques. They seem to result in similar graft survival, but differ in maximum storage time, technical aspects, tissue evaluation possibilities and microbiological safety. Organ culture seems to offer several advantages, yet the use of commercial fetal calf serum gives concern regarding the possible transfer of unknown microbiological agents. Finally, recent advantages in preservation of cultured corneal/limbal epithelia may increase the availability of this tissue and therefore increase treatment of limbal stem cell deficiency. It remains to be shown whether further improvements in storage methology also can bring other fields of regenerative medicine forward by improving the availability and safety of transplantation of cultured cells