In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP-NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a k(cat) of 16 s(-1) and a K(m) of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn-Zn distance of 3.38 Å. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA.

In an effort to probe for metal binding to metallo-β-lactamase (MβL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.

Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCLdependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immunebased NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.is one of the most abundant nonribosomal proteins in the nucleolus (1), first identified in ribosomal RNA processing (2). Additional studies have demonstrated that NCL is a multifunctional nucleocytoplasmic protein, involved in ribosomal assembly, chromatin decondensation, transcription, nucleo-cytoplasmic import/export, and chromatin remodeling (3, 4). NCL is frequently up-regulated in cancer and in cancerassociated endothelial cells compared with normal tissues (5, 6), where it is also present on the cell surface (7,8). Altered NCL expression and localization results in oncogenic effects, such as stabilization of AKT, Bcl-2, Bcl-XL, and IL-2 mRNAs (9-11). Moreover, surface-NCL acts as a receptor for several oncogenic ligands (12-15) and viruses (16). Recently, we reported that NCL has a critical protumorigenic function regulating the biogenesis of selected microRNAs (miRNAs), a class of noncoding single-stranded RNAs 19-22 nt in length (17) that regulate gene expression at the posttranscriptional level by targeting mRNAs in a sequence-specific manner (18). In fact, NCL enhances the maturation of specific miRNAs (including miR-21, miR-221, and miR-222) causally involved in cancer pathogenesis and resistance to several antineoplastic treatments (19-23). Our findings demonstrated that NCL modulates the biogenesis of these miRNAs at the posttranscriptional level, enhancing their maturation from pri-to premiRNAs, identifying a novel NCL-dependent oncogenic mechanism (19).Because of its oncogenic role and specific expression on cancer cells surface, NCL represents an attractive target f...

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive.We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis.In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

The metallo-β-lactamases (MβLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in β-lactam-containing antibiotics, and render the antibiotics inactive. All known MβLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MβL L1 with spin labels at position 163 and 286 analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was “diluted” by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling.

Rationale: miR-155 is a pro-inflammatory microRNA upregulated in human and mouse macrophages exposed to lipopolysaccharide (LPS) that is required to mount an effective immune response. High levels of miR-155 are observed in different solid tumors as well as leukemias, including chronic lymphocytic leukemia (CLL). Quaking (QKI) is a tumor-suppressor gene encoding a conserved RNA-binding protein. In silico analyses predict that the QKI transcript is a target of miR-155, and we hypothesized that miR-155 might carry out its pro-inflammatory and oncogenic signals at least in part by targeting QKI. Methods: Mouse RAW-264.7 macrophages were stimulated with LPS or mock PBS three times over a period of 6 days. qRT-PCR was used to monitor the expression of QKI, miR-155 and Tnf (tumor necrosis factor alpha), and Quaking protein (Qki) expression was analyzed by Western blot. Expression of miR-155 and QKI in Burkitt's lymphoma cell lines and CLL cell lines was measured by qRT-PCR; QKI expression was also determined by Western blot. Cell lines were transfected with miR-155 or miR-control, or 155-I, and Western blot analysis of QKI was performed after 48 hours. To study the in vivo effects of the cooperation between miR-155 and QKI, we analyzed Qki expression in splenic B cells from wild type C57B1/6 mice, miR-155−/- mice, and Eμ-miR-155 transgenic mice (which develop aggressive CLL), with and without LPS challenge. Finally, expression of Qki was determined in samples from patients with B-cell CLL. Results: After 8 hours of LPS challenge there was a 2-fold decrease in QKI expression, while miR-155 and Tnf both increased approximately 10-fold (p<0.05). QKI returned to pre-treatment levels at 2 days, while miR-155remained high for 3 days. LPS re-stimulation at 3 days (mimicking chronic inflammation) reduced QKI expression 2.3-fold (p<0.05) over a 48 hour period in parallel with a renewed up-regulation of miR-155, and Western blotting confirmed the above observed changes for Qki. CLL-derived cell lines showed significantly higher expression of miR-155, and lower expression of QKI, compared with Burkitt's lymphoma cell lines (p<0.05). Transfection of miR-155 led to decreased Qki expression in Burkitt's lymphoma lines, while 155-I transfection of MEC2 CLL cells caused Qki upregulation at 48 hours. Splenic B cells from leukemic Eμ-miR-155 mice had decreased Qki levels compared with C57B1/6 mice and miR-155−/−mice, and LPS challenge of cultured B cells resulted in a pronounced downregulation of Qki expression only in B cells coming from Eμ-miR-155 mice. Finally, reduced expression of Qki was observed in B cells coming from patients with CLL compared with healthy patients. Conclusions: The inverse relationship between miR-155 and Qki is specific, and may represent a regulatory mechanism for the immune response mounted by macrophages and B cells. This relationship also sheds light on the pro-inflammatory and oncogenic properties of miR-155 in leukemia. Citation Format: Timothy K. Richmond, Esmerina Tili, Melissa Brown, Marcela Chiabai, Dario Palmieri, Ri Cui, Tyler Sheetz, Carlo M. Croce. Interaction between miR-155 and Quaking in the innate immune response and leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-289. doi:10.1158/1538-7445.AM2015-LB-289

Currently, most of the anti-neoplastic treatments for lung cancer patients are based on DNA-damaging cytotoxic therapies such as platinum-containing compounds and radiotherapy. Therefore, a better understanding of the molecular mechanisms involved in the cellular response to genotoxic stresses is required in order to improve the clinical management of lung cancer. Ran Binding Protein 9 (RanBP9, also known as RanBP-M) is a ubiquitous and evolutionary conserved scaffold protein that shuttles between the nucleus and the cytoplasm, and interacts with many major players in tumor biology. However, its biological functions are not well studied and still debated. Here, we show that RanBP9 interacts with and is phosphorylated by ATM, one of the apical activators of the DNA damage response (DDR) following DNA Double-Strand Breaks (DSBs). Following ATM-dependent phosphorylation, RanBP9 rapidly accumulates into the nucleus of lung cancer cells. By using three different lung cancer cell lines (A549, H460, and H1299), we found that stable silencing of RanBP9 (ShRanBP9) significantly affects the DDR. In fact, stable ShRanBP9 clones display a delayed and/or reduced activation of key components of the cellular response to Ionizing Radiation (IR), including ATM, Chk2, H2AX-γ and p53. Accordingly, abrogation of RanBP9 expression significantly affected Homology-Directed repair of damaged DNA. On the other hand, stable silencing of RanBP9 results in increased IR-induced senescence and apoptosis. In summary, here we present evidence that RanBP9 is a novel mediator of the cellular DDR, whose recruitment into the nucleus upon IR is dependent on ATM kinase activity. In turn, nuclear RanBP9 participates to the efficient activation of cellular DDR. On the contrary, its absence hampers the repair of damaged DNA following DSBs, resulting in enhanced lung cancer sensitivity to genotoxic stresses. Taken together, our findings suggest that targeting RanBP9 might represent a new potential approach to increase sensitivity of lung cancer cells to genotoxic anti-neoplastic treatments. Citation Format: Dario Palmieri, Mario Scarpa, Anna Tessari, Rexhep Uka, Foued Amari, Cindy Lee, Timothy Richmond, Tyler Sheetz, Jeffrey Parvin, Thomas Ludwig, Carlo M. Croce, Vincenzo Coppola. Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-164. doi:10.1158/1538-7445.AM2015-LB-164

No abstract