Challenging packaging limits and infectivity of phage λ

The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet these motors are hindered at the end of the packaging process by the progressive build-up of a force resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage λ and still yield infectious virions, and the conditions under which this can be efficiently performed. Using a packaging strategy developed in our laboratory of building phage λ from scratch, together with plaque assay monitoring, we have been able to show that the terminase motor of phage λ is able to produce infectious particles with up to 110% of the wild-type (WT) λ-DNA length. However, the phage production rate, and thus the infectivity, decreased exponentially with increasing DNA length, and was a factor of 103 lower for the 110% λ-DNA phage. Interestingly, our in vitro strategy was still efficient in fully packaging phages with DNA lengths as high as 114% of the WT length, but these viruses were unable to infect bacterial cells efficiently. Further, we demonstrated that the phage production rate is modulated by the presence of multivalent ionic species. The biological consequences of these finding are discussed

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis.

Inhibitors of mitochondrial respiration and ATP synthesis may promote the selective killing of respiration-competent cancer cells that are critical for tumor progression. We previously reported that CADD522, a small molecule inhibitor of the RUNX2 transcription factor, has potential for breast cancer treatment. In the current study, we show that CADD522 inhibits mitochondrial oxidative phosphorylation by decreasing the mitochondrial oxygen consumption rate (OCR) and ATP production in human breast cancer cells in a RUNX2-independent manner. The enzyme activity of mitochondrial ATP synthase was inhibited by CADD522 treatment. Importantly, results from cellular thermal shift assays that detect drug-induced protein stabilization revealed that CADD522 interacts with both α and β subunits of the F1-ATP synthase complex. Differential scanning fluorimetry also demonstrated interaction of α subunits of the F1-ATP synthase to CADD522. These results suggest that CADD522 might target the enzymatic F1 subunits in the ATP synthase complex. CADD522 increased the levels of intracellular reactive oxygen species (ROS), which was prevented by MitoQ, a mitochondria-targeted antioxidant, suggesting that cancer cells exposed to CADD522 may elevate ROS from mitochondria. CADD522-increased mitochondrial ROS levels were enhanced by exogenously added pro-oxidants such as hydrogen peroxide or tert-butyl hydroperoxide. Conversely, CADD522-mediated cell growth inhibition was blocked by N-acetyl-l-cysteine, a general ROS scavenger. Therefore, CADD522 may exert its antitumor activity by increasing mitochondrial driven cellular ROS levels. Collectively, our data suggest in vitro proof-of-concept that supports inhibition of mitochondrial ATP synthase and ROS generation as contributors to the effectiveness of CADD522 in suppression of tumor growth

Allosteric inhibitor of β-catenin selectively targets oncogenic Wnt signaling in colon cancer.

Abnormal regulation of β-catenin initiates an oncogenic program that serves as a main driver of many cancers. Albeit challenging, β-catenin is an attractive drug target due to its role in maintenance of cancer stem cells and potential to eliminate cancer relapse. We have identified C2, a novel β-catenin inhibitor, which is a small molecule that binds to a novel allosteric site on the surface of β-catenin. C2 selectively inhibits β-catenin, lowers its cellular load and significantly reduces viability of β-catenin-driven cancer cells. Through direct binding to β-catenin, C2 renders the target inactive that eventually activates proteasome system for its removal. Here we report a novel pharmacologic approach for selective inhibition of β-catenin via targeting a cryptic allosteric modulation site. Our findings may provide a new perspective for therapeutic targeting of β-catenin

Structural and Functional Studies of Wilms Tumour Protein - WT1; Novel Insights into Functionality of Zinc Finger Proteins

The molecular process under which message in DNA is relayed onto RNA is called transcription, and it accomplished through involvement of so-called transcription factors. Zinc finger proteins are one of the most abundant classes of regulatory proteins in eukaryotic cells where they play central roles in a variety of cellular activities. A classical zinc finger is a small protein motif with a simple beta-beta-alpha secondary structure stabilized by a zinc ion that is coordinated by two conserved cysteines and two histidines. Wilms tumour protein (WT1) is a transcription factor, which in normal cells regulates a vast network of genes during development of the kidney and the genitourinary system; its malfunction may lead to serious abnormalities such as WAGR syndrome, Denys Drash syndrome and Frasier syndrome. WT1 has been interesting for researchers as a zinc finger protein, and as a transcription factor having both oncogenic and tumor suppressor functions. WT1 contains four C2H2-type zinc fingers and it specifically binds to GC-rich sequences in the promoter regions of its target genes, which are either up- or down-regulated. It has a dual functionality: it can bind specifically to DNA and RNA. Alternative splicing of the immature WT1 mRNA at exon 5 and exon 9 produces 4 distinct isoforms with 17 and 3 amino acids (KTS) insertions, respectively. The KTS insertion into the linker between zinc fingers 3 and 4 abrogates binding to the DNA. On the other hand, it differentially affects affinity of WT1 for its single-stranded RNA targets ? this is not yet understood. Thus, two unique features of WT1 differentiate it from other zinc finger proteins of the same class: the KTS insertion and an unconventional amino acid composition of its zinc finger 1. We have undertaken an interdisciplinary approach in order to better understand the nucleic acid-binding features of WT1. In the Paper I we describe a study where we express, purify the WT1 protein and make its biochemical characterization. In Paper II a surface plasmon resonance-based study addresses the DNA-binding properties of WT1. Paper III describes an analogous study where RNA-binding properties of WT1 are investigated. Finally, Paper IV is a Bacterial 1-Hybrid System-based study that is directed towards elucidation of the DNA-binding preferences of zinc finger 1. Interesting conclusions about the functionality of the KTS insertion and the zinc finger 1 are reached

Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1

Wilm's Tumor gene 1 (WT1) encodes a zinc finger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-finger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN-(wt1) and 6HIS-ZN+(wt1), corresponding to two alternative splice variants of the C-terminal regions of human WT1 (-KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni2+-NTA affinity and size-exclusion chromatography was applied for purification of the proteins in denaturing conditions. The effects of various buffers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected beta beta alpha content for the refolded proteins, with a notable degradation of the alpha-helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5'-GCG-T GG-GCG-3' confirmed that 6HIS-ZN-(wt1) has higher DNA binding affinity than 6HIS-ZN+(wt1). (c) 2005 Elsevier Inc. All rights reserved

Thrombospondin-1 counteracts the p97 inhibitor CB-5083 in colon carcinoma cells.

p97 has recently emerged as a therapeutic target for cancer due to its essential functions in protein homeostasis. CB-5083 is a first-in-class, potent and selective ATP-competitive p97 inhibitor that induces proteotoxic stress in cancer cells. Potential mechanisms regulating the sensitivity of cells to p97 inhibition remain poorly studied. Here, we demonstrate that Thrombospondin-1 (THBS1) is a CB-5083-upregulated gene that helps confer resistance of HCT116 cells to CB-5083. Our immunoblotting and immunofluorescence data showed that CB-5083 significantly increases the steady-state abundance of THBS1. Blockade of THBS1 induction sensitized cells to CB-5083-mediated growth inhibition. Suppression of THBS1 caused an increase of CB-5083-induced sub-G1 population and caspase 3/7 activity suggesting that its function is linked to the survival of cancer cells in response to p97 inhibition. Altogether our data provide new evidence that THBS1 is important for the susceptibility of cells to p97 inhibition

Kinetic behaviour of WT 1's zinc finger domain in binding to the alpha-actinin-1 mRNA

The zinc finger transcription factor Wilms tumour protein (WT 1) is known for its essential involvement in the development of the genitourinary system as well as of other organs and tissues. WT 1 is capable of selectively binding either DNA or mRNA targets. A KTS insertion due to alternative splicing between the zinc fingers 3 and 4 and an unconventional zinc finger 1 are the unique features that distinguish WT 1 from classical DNA-binding C2H2-type zinc finger proteins. The DNA binding characteristics of WT 1 are well studied. Due to lack of information about its native RNA targets, no extensive research has been directed at how WT 1 binds RNA. Using surface plasmon resonance, this study attempts to understand the binding behaviour of WT 1 zinc fingers with its recently reported and first putative mRNA target, ACT 34, whose stem-loop structure is believed to be critical for the interactions with WT 1. We have analysed the interactions of five WT 1 zinc finger truncations with wild-type ACT 34 and four variants. Our results indicate that WT 1 zinc fingers bind ACT 34 in a specific manner, and that this occurs as interplay of all four zinc fingers. We also report that a sensitive kinetic balance, which is equilibrated by both zinc finger 1 and KTS, regulates the interaction with ACT 34. The stem-loop and the flanking nucleotides are important elements for specific recognition by WT 1 zinc fingers. © 2010 Elsevier Inc