No abstract

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra-and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.Integration of the reverse-transcribed RNA genome into a host chromosome is an obligatory step for HIV-1 3 replication (reviewed in Ref. 1). This process is catalyzed by the retroviral enzyme integrase (IN) in two reaction steps. In the first step, which is called 3Ј-processing and takes place shortly after the cDNA is made, IN hydrolyzes a GT dinucleotide from each end of the viral DNA. In the second step, IN catalyzes concerted integration of the processed viral DNA ends into chromosomal DNA. The sites of attack on the two target DNA strands are separated by 5 bp, which leads to dissociation of the small double-stranded DNA fragment between the attachment sites. The subsequent repair of the intermediate species by cellular enzymes completes the integration reaction. HIV-1 IN consists of three distinct structural and functional domains. The N-terminal domain (NTD) (residues 1-50) contains conserved pairs of histidine and cysteine residues that bind zinc (2, 3), which contributes to IN multimerization and its catalytic function (4, 5). The catalytic core domain (CCD) (residues 51-212) contains three acidic residues, Asp-64, Asp-116, and Glu-152, which play a key role in coordinating active site divalent metal ions (6, 7). The C-terminal domain (CTD) (residues 213-288) also contributes to functional IN multimerization (8, 9). Results of structural biology studies revealed each individual domain as a dimer (3,6,7,10,11) and more recent two-domain crystal structures comprised of the CCD and CTD (12) or NTD and CCD (13)...

The Wnt/b-catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45-mutated-b-catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated b-catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin-D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2-fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S-phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of b-catenin along with an increase in total and nuclear cyclin-D1 protein at 40 hours in TG livers. To test if stimulation of b-catenin induces regeneration, we used hydrodynamic delivery of Wnt-1 naked DNA to control mice, which prompted an increase in Wnt-1, b-catenin, and known targets, glutamine synthetase (GS) and cyclin-D1, along with a concomitant increase in cell proliferation. b-Catenin-overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed upregulation of b-catenin, cyclin-D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. Conclusion: b-Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, b-catenin overexpression makes TG mice susceptible to DEN-induced HCC. (HEPATOLOGY 2010;51:1603-1613 W nt/b-catenin signaling is an evolutionarily well-conserved pathway and important in liver health and repair.1 In adult liver, bcatenin signaling is essentially quiescent, with active bcatenin restricted to hepatocytes in the centrizonal area where it regulates expression of genes such as glutamine synthetase (GS) and others involved in xenobiotic metabolism.2 In other hepatocytes, b-catenin steady state is achieved by phosphorylation at key serine/threonine residues and subsequent degradation, and is predominantly localized to membrane to mediate cell-cell adhesion by forming a bridge between E-cadherin and actin cytoskeleton. Activation of b-catenin signaling during liver regeneration has been reported in rats and mice. [4][5][6][7] Although a positive regulator in the activity of normal liver growth, aberrant activation of the Wnt/bcatenin pathway is implicated in hepatocarcinogenesis,