Michael J. Haykinson

The Hin dimer interface is critical for Fis-mediated activation of the catalytic steps of site-specific DNA inversion

BACKGROUND Hin is a member of an extended family of site-specific recombinases--the DNA invertase/resolvase family--that catalyze inversion or deletion of DNA. DNA inversion by Hin occurs between two recombination sites and requires the regulatory protein Fis, which associates with a cis-acting recombinational enhancer sequence. Hin recombinase dimers bind to the two recombination sites and assemble onto the Fis-bound enhancer to generate an invertasome structure, at which time they become competent to catalyze DNA cleavage and strand exchange. In this report, we investigate the role of the Hin dimer interface in the activation of its catalytic functions. RESULTS We show that the Hin dimer is formed at an interface that contains putative amphipathic alpha-helices in a manner that is very similar to gamma delta resolvase. Certain detergents weakened cooperative interactions between the subunits of the Hin dimer and dramatically increased the rate of the first chemical step of the reaction--double-strand cleavage events at the center of the recombination sites. Amino-acid substitutions within the dimer interface led to profound changes in the catalytic properties of the recombinase. Nearly all mutations strongly affected the ability of the dimer to cleave DNA and most abolished DNA strand exchange in vitro. Some amino-acid substitutions altered the concerted nature of the DNA cleavage events within both recombination sites, and two mutations resulted in cleavage activity that was independent of Fis activation in vitro. Disulfide-linked Hin dimers were catalytically inactive; however, subsequent to the addition of the Fis-bound enhancer sequence, catalytic activity was no longer affected by the presence of oxidizing agents. CONCLUSIONS The combined results demonstrate that the Hin dimer interface is of critical importance for the activation of catalysis and imply that interactions with the Fis-bound enhancer may trigger a conformational adjustment within the region that is important for concerted DNA cleavage within both recombination sites, and possibly for the subsequent exchange of DNA strands.

The site-specific integration reaction of Listeria phage A118 integrase, a serine recombinase

BackgroundA large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain. Members of this subfamily often function as phage integrases but also mediate transposition and regulate terminal differentiation processes in eubacteria. Although a few members of this subfamily have been studied in purified in vitro systems, key mechanistic aspects of reactions promoted by these recombinases remain to be determined, particularly with respect to the functions of the large C-terminal domain.ResultsWe have developed and characterized a robust in vitro recombination reaction by the Listeria phage A118 integrase, a member of the subfamily of serine recombinases containing a large C-terminal domain. The reaction occurs in a simple buffered salt solution and exhibits a modest stimulation by divalent cations or spermidine and DNA supercoiling. Recombination with purified A118 integrase is unidirectional, being efficient only between attP and attB DNA sites to either join separate DNA molecules (intermolecular recombination) or to generate deletions or inversions depending on the relative orientation of att sites in cis (intramolecular recombination). The minimal attP site is 50 bp but requires only 44 bp of base sequence information, whereas the minimal attB site is 42 bp and requires 38 bp of base sequence information. DNA exchange occurs between the central 2 bp of attP and attB. Identity between these two base pairs is required for recombination, and they solely determine the orientation of recombination sites. The integrase dimer binds efficiently to full att sites, including the attL and attR integration products, but poorly and differentially to each half-site. The large C-terminal domain can be separated from the N-terminal catalytic by partial proteolysis and mediates non-cooperative DNA binding to att sites.ConclusionsThe basic properties of the phage A118 integrase reaction and its substrate requirements have been elucidated. A118 integrase thus joins the handful of biochemically characterized serine integrases that are serving as models for mechanistic studies on this important class of recombinases. Information reported here will also be useful in exploiting this recombinase for genetic engineering.

Amplification Target ADRM1: Role as an Oncogene and Therapeutic Target for Ovarian Cancer

Approximately 25,000 ovarian cancers are diagnosed in the U.S. annually, and 75% are in the advanced stage and largely incurable. There is critical need for early detection tools and novel treatments. Proteasomal ubiquitin receptor ADRM1 is a protein that is encoded by the ADRM1 gene. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. Its overexpression correlated significantly with shorter time to recurrence and overall survival. Array-CGH and microarray expression of ovarian cancer cell lines provided evidence consistent with primary tumor data that ADRM1 is a 20q13 amplification target. Herein, we confirm the ADRM1 amplicon in a second ovarian cancer cohort and define a minimally amplified region of 262 KB encompassing seven genes. Additionally, using RNAi knock-down of ADRM1 in naturally amplified cell line OAW42 and overexpression of ADRM1 via transfection in ES2, we show that (1) ADRM1 overexpression increases proliferation, migration, and growth in soft agar, and (2) knock-down of ADRM1 results in apoptosis. Proteomic analysis of cells with ADRM1 knock-down reveals dysregulation of proteins including CDK-activating kinase assembly factor MAT1. Taken together, the results indicate that amplified ADRM1 is involved in cell proliferation, migration and survival in ovarian cancer cells, supporting a role as an oncogene and novel therapeutic target for ovarian cancer.

HU and functional analogs in eukaryotes promote Hin invertasome assembly.

The prokaryotic protein HU functions as an accessory factor in many different biochemical reactions. We have characterized the role of HU in assembling the invertasome, an intermediate nucleoprotein complex involved in Hin-mediated site-specific recombination. Formation of this complex requires the looping of intervening DNA segments between sites bound by the Hin recombinase and the Fis protein. HU stimulates this process on substrates containing intervening segments of length < 100 bp. Characterization of the activity of HU in Hin-mediated recombination in vitro and in vivo yields evidence that its role in this reaction is primarily to facilitate the looping of the intervening DNA segment. By using this reaction as an assay, we identify proteins from mammals, yeast, trypanosomes, and wheat which can fulfill the same function in vitro. Using ligase-mediated circularization of short DNA fragments we also show that HU, the high mobility group (HMG) 1 and 2 proteins from mammals, and a protein from yeast can bend DNA extremely efficiently. These results support the view that this ubiquitous class of proteins enhance the assembly of nucleoprotein complexes under conditions of limited DNA flexibility.

Proteomic identification of mitochondrial targets of arginase in human breast cancer.

We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by Nω hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA’s action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.

The E3 ubiquitin ligase TRIM32 regulates myoblast proliferation by controlling turnover of NDRG2

Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32s substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation.

Novel Cell and Tissue Acquisition System (CTAS): Microdissection of Live and Frozen Brain Tissues

We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS - Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

C-terminally truncated form of αB-crystallin is associated with IDH1 R132H mutation in anaplastic astrocytoma

Malignant gliomas are the most common human primary brain tumors. Point mutation of amino acid arginine 132 to histidine (R132H) in the IDH1 protein leads to an enzymatic gain-of-function and is thought to promote gliomagenesis. Little is known about the downstream effects of the IDH1 mutation on protein expression and how and whether changes in protein expression are involved in tumor formation or propagation. In the current study, we used 2D DIGE (difference gel electrophoresis) and mass spectrometry to analyze differences in protein expression between IDH1R132H mutant and wild type anaplastic (grade III) astrocytoma from human brain cancer tissues. We show that expression levels of many proteins are altered in IDH1R132H mutant anaplastic astrocytoma. Some of the most over-expressed proteins in the mutants include several forms of αB-crystallin, a small heat-shock and anti-apoptotic protein. αB-crystallin proteins are elevated up to 22-fold in IDH1R132H mutant tumors, and αB-crystallin expression appears to be controlled at the post-translational level. We identified the most abundant form of αB-crystallin as a low molecular weight species that is C-terminally truncated. We also found that overexpression of αB-crystallin can be induced by transfecting U251 human glioblastoma cell lines with the IDH1R132H mutation. In conclusion, the association of a C-terminally truncated form of αB-crystallin protein with the IDH1R132H mutation is a novel finding that could impact apoptosis and stress response in IDH1 mutant glioma.

Abstract 1210: Activation of heterogeneous nuclear ribonucleoprotein K (HNRNPK) by ERBB2/ERBB3

ERBB2 (HER2/neu) amplification constitute one of the most prominent oncogenic amplification event in breast cancer, and ERBB2 overexpression is well established as a predominantly proliferation and metastasis favoring signaling input. It is consequently the object of different targeted therapies (e.g. Trastuzumab, Pertuzumab or Lapatinib). We carried out 2-D difference gel electrophoresis based proteomics studies (DIGE) of early posttranslational protein modifications in MCF7 breast cancer cells in response to ligand induced activation. These studies have identified heterogeneous nuclear ribonucleoprotein K (HNRNPK) as a target for rapid tyrosine phosphorylation after ERBB2/ERBB3 activation by neuregulin β1. Upregulation of protein levels coupled with cytoplasmic accumulation of HNRNPK has been found to be associated with its oncogenic role in several types of cancer, and HNRNPK overexpression enhances the tumorigenicity of breast cancer cells in cell culture models. Previous studies have shown that HNRNPK levels are reduced in breast cancer cells after prolonged treatment with EGFR or ERBB2 directed therapeutic antibodies. However, while molecular mechanistic data exist on HNRNPK from a variety of model systems, we currently lack a coherent mechanistic framework of the crosstalk of ERBB2 and HNRNPK signaling in breast cancer. Following our DIGE analysis, we found that ligand induced and ERBB2/ERBB3 mediated tyrosine phosphorylation of HNRNPK in MCF7 cells is mediated by members of the SRC kinase family. Following ligand dependent stimulation, activation of HNRNPK coincides with a cytoplasmic relocalization of a relatively small portion of the predominantly nuclear HNRNPK pool. We compared our observations of ligand induced activation with the status of HNRNPK in ERBB2 over expressing breast cancer cell lines in presence or absence of ERBB2 targeted inhibitors. These studies are aimed at elucidating the interplay of HNRNPK and constitutive ERBB2 signaling in breast cancer cells and its role in cellular transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1210. doi:10.1158/1538-7445.AM2011-1210

404 Nω-HYDROXY L-ARGININE-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS: PROTEOMIC APPROACH FOR THE IDENTIFICATION OF KEY TARGET PROTEINS.

N Ω -Hydroxy l-arginine (NOHA) induces apoptosis in human breast cancer MDA-MB-468 cells by caspase 8-mediated cleavage of proapoptotic Bid molecule, leading to the release of cytochrome c and activation of caspase 3. This apoptotic action was antagonized by exogenous l-ornithine (Singh et al. Cancer Research 2000; Singh et al. J Biol Chem 2002). Objective To identify key proteins involved during NOHA-induced apoptosis and understand precise mechanisms by which l-ornithine inhibit NOHA-induced apoptosis. Methods and Results We transfected MDA-MB-468 cells with full-length Bcl2 and selected stable cells line overexpressing Bcl2. NOHA-induced cytochrome c release, activation of caspase 3, and inhibition of cell proliferation were blocked in these cells; however, it did not blocked cleavage of Bid or caspase-8 activation, suggesting that mitochondria may be the primary site of NOHA action. We performed proteomic analysis of control cells and cells treated with NOHA (1 mM) alone or in combination with l-ornithine (0.5 mM). Protein samples obtained from all three groups were labeled with Cy2-, Cy3-, and Cy5-based fluorescent dyes and analyzed using methods of difference gel electrophoresis (DIGE). Samples were subjected to two-dimensional electrophoresis and fluorescent images were obtained. More than 100 spots have been identified as having protein expression changes of 1.5 times or more after NOHA treatment compared with a control group. The majority of proteins after simultaneous treatment with l-ornithine and NOHA had very similar protein expression profiles as a control group. Proteins of interest are currently being excised from gels, digested with trypsin, and identified using a MALDI TOF-TOF mass spectrometer. Significance Macrophages and myoepithelial cells at the site of breast cancer express nitric oxide synthase (NOS) and therefore have the capacity to generate both NOHA and NO. Proteomic analysis leading to the identification of key mitochondrial proteins targeted by these two signaling molecules, during macrophage activation, as a result of various inflammatory diseases including breast cancer, will help our overall understanding of the process required for better therapeutic interventions.