Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy

The design, synthesis and evaluation of four novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) prodrugs (1a,b and 2a,b; ) for potential use in carboxypeptidase G2 (CPG2)-based antibody-directed enzyme prodrug therapy (ADEPT) is reported. Although all four prodrugs were shown to be less cytotoxic than the released parent PBDs 3 and 4, the urea prodrugs 1b and 2b were found to be too unstable for use in ADEPT, whereas carbamates 1a and 2a are both stable in an aqueous environment and are good substrates for CPG2.     

Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals

A recent study suggests that the opportunistic pathogen Pseudomonas aeruginosa can actively monitor the host immune system. The P. aeruginosa outer membrane protein OprF was found to bind specifically to the cytokine interferon-gamma (IFN-gamma), and this interaction upregulated production of virulence factors through a cell-cell communication system known as quorum sensing (QS). Taken together with previous findings that P. aeruginosa QS can alter the host immune response (e.g. by activation of IFN-gamma), these data illustrate an exciting new element of bacteria-host interactions in which the P. aeruginosa quorum-sensing system both senses and modulates the host immune state.     

Non-coding RNA in fly dosage compensation

Dosage compensation modulates global expression of an X chromosome and is necessary to restore the balance between X-chromosome and autosome expression in both sexes. A central question in the field is how this regulation is directed. Large non-coding RNAs, such as Xist in mammals and roX in flies, have pivotal roles in targeting chromosome-wide modification for dosage compensation. Several recent studies in Drosophila provide new insight into the principles of X-chromosome recognition and the function of non-coding RNA in this process.     

Quaternary structure of a SPATE autotransporter protein

The temperature-sensitive hemagglutinin (Tsh) is a representative of the growing subfamily of secreted bacterial virulence factors, known as serine protease autotransporters of the Enterobacteriaceae (SPATEs). Expressed by avian and human pathogenic strains of Escherichia coli Tsh acts as a serine protease and an adhesin to erythrocytes, hemoglobin, and extracellular matrix proteins. Mature Tsh is comprised of a 106-kDa secreted domain (Tshs) and a 33-kDa outer membrane beta-domain (Tshbeta). Based on the size of beta-domains and functional properties of their passenger domains, all SPATEs are considered to be conventional autotransporters. However, it is unsettled if the conventional autotransporters exist as monomers, oligomers, or multimers (e.g., hexamers). To determine the quaternary structure of Tsh in vitro, we purified Tshbeta from the outer membranes and showed that it is natively folded because it is heat modifiable and resistant to protease digestion. Blue-native polyacrylamide gel electrophoresis of Tshbeta indicated that Tshbeta exists as a monomer or a dimer. The cross-linking analysis demonstrated that purified Tshbeta exists as a monomer. The size-exclusion chromatography and cross-linking analyses of purified Tshs also showed that the passenger domain of Tsh is a monomer. Overall, our data indicated that Tsh is a monomeric protein in vitro and support the concept that the SPATE autotransporters exist as monomers rather than as multimers. Implications of our findings on the mechanism of autotransporter secretion across the outer membrane are discussed.     

Influence of chromatin and single strand binding proteins on the activity of an archaeal MCM

The mini-chromosome maintenance (MCM) complex is the presumptive replicative helicase in archaea and eukaryotes. In archaea, the MCM is a homo-multimer, in eukaryotes a heterohexamer composed of six related subunits, MCM 2-7. Biochemical studies using naked DNA templates have revealed that archaeal MCMs and a sub-complex of eukaryotic MCM 4, 6 and 7 have 3' to 5' helicase activity. Here, we investigate the influence of the major chromatin proteins, Alba and Sul7d, of Sulfolobus solfataricus (Sso) on the ability of the MCM complex to melt partial duplex DNA substrates. In addition, we test the effect of Sso SSB on MCM activity. We reveal that Alba represents a formidable barrier to MCM activity and further demonstrate that acetylation of Alba alleviates repression of MCM activity.     

Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3

The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.     

Tracking of peptide-specific CD4+ T-cell responses after an acute resolving viral infection: a study of parvovirus B19

The evolution of peptide-specific CD4(+) T-cell responses to acute viral infections of humans is poorly understood. We analyzed the response to parvovirus B19 (B19), a ubiquitous and clinically significant pathogen with a compact and conserved genome. The magnitude and breadth of the CD4(+) T-cell response to the two B19 capsid proteins were investigated using a set of overlapping peptides and gamma interferon-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells (PBMCs) from a cohort of acutely infected individuals who presented with acute arthropathy. These were compared to those for a cohort of B19-specific immunoglobulin M-negative (IgM(-)), IgG(+) remotely infected individuals. Both cohorts of individuals were found to make broad CD4(+) responses. However, while the responses following acute infection were detectable ex vivo, responses in remotely infected individuals were only detected after culture. One epitope (LASEESAFYVLEHSSFQLLG) was consistently targeted by both acutely (10/12) and remotely (6/7) infected individuals. This epitope was DRB1*1501 restricted, and a major histocompatibility complex peptide tetramer stained PBMCs from acutely infected individuals in the range of 0.003 to 0.042% of CD4(+) T cells. Tetramer-positive populations were initially CD62L(lo); unlike the case for B19-specific CD8(+) T-cell responses, however, CD62L was reexpressed at later times, as responses remained stable or declined slowly. This first identification of B19 CD4(+) T-cell epitopes, including a key immunodominant peptide, provides the tools to investigate the breadth, frequency, and functions of cellular responses to this virus in a range of specific clinical settings and gives an important reference point for analysis of peptide-specific CD4(+) T cells during acute and persistent virus infections of humans.     

Suppression of proinflammatory signal transduction and gene expression by the dual nucleic acid binding domains of the vaccinia virus E3L proteins

Cells have evolved elaborate mechanisms to counteract the onslaught of viral infections. To activate these defenses, the viral threat must be recognized. Danger signals, or pathogen-associated molecular patterns, that are induced by pathogens include double-stranded RNA (dsRNA), viral single-stranded RNA, glycolipids, and CpG DNA. Understanding the signal transduction pathways activated and host gene expression induced by these danger signals is vital to understanding virus-host interactions. The vaccinia virus E3L protein is involved in blocking the host antiviral response and increasing pathogenesis, functions that map to separate C-terminal dsRNA- and N-terminal Z-DNA-binding domains. Viruses containing mutations in these domains allow modeling of the role of dsRNA and Z-form nucleic acid in the host response to virus infection. Deletions in the Z-DNA- or dsRNA-binding domains led to activation of signal transduction cascades and up-regulation of host gene expression, with many genes involved in the inflammatory response. These data suggest that poxviruses actively inhibit cellular recognition of viral danger signals and the subsequent cellular response to the viral threat.     

His-384 allotypic variant of factor H associated with age-related macular degeneration has different heparin binding properties from the non-disease-associated form

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.