Aberrant Activity of Histone-Lysine N-Methyltransferase 2 (KMT2) Complexes in Oncogenesis.

KMT2 (histone-lysine N-methyltransferase subclass 2) complexes methylate lysine 4 on the histone H3 tail at gene promoters and gene enhancers and, thus, control the process of gene transcription. These complexes not only play an essential role in normal development but have also been described as involved in the aberrant growth of tissues. KMT2 mutations resulting from the rearrangements of the KMT2A (MLL1) gene at 11q23 are associated with pediatric mixed-lineage leukemias, and recent studies demonstrate that KMT2 genes are frequently mutated in many types of human cancers. Moreover, other components of the KMT2 complexes have been reported to contribute to oncogenesis. This review summarizes the recent advances in our knowledge of the role of KMT2 complexes in cell transformation. In addition, it discusses the therapeutic targeting of different components of the KMT2 complexes.

Substrate-assisted catalysis by PARP10 limits its activity to mono-ADP-ribosylation.

ADP-ribosylation controls many processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARP6-PARP16, raising questions about how these enzymes function. We find that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has transferase rather than polymerase activity. Despite this fundamental difference, PARP10 also modifies acidic residues. Consequently, we propose an alternative catalytic mechanism for PARP10 compared to PARP1 in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate by using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells.

The plant s1-like nuclease family has evolved a highly diverse range of catalytic capabilities.

Plant S1-like nucleases, often referred to as nuclease I enzymes, are the main class of enzymes involved in nucleic acid degradation during plant programmed cell death. The catalytically active site of these enzymes shows a significant similarity to the well-described P1 nuclease from Penicillium citrinum. Previously published studies reported that plant S1-like nucleases possess catalytic activities similar to their fungal orthologs, i.e. they hydrolyze single-stranded DNA and RNA, and less efficiently double-stranded DNA, in the presence of zinc ions. Here we describe a comprehensive study of the nucleolytic activities of all Arabidopsis S1-like paralogs. Our results revealed that different members of this family are characterized by a surprisingly large variety of catalytic properties. We found that, in addition to Zn(2+)-dependent enzymes, this family also comprises nucleases activated by Ca(2+) and Mn(2+), which implies that the apparently well-known S1 nuclease active site in plant nucleases is able to cooperate with different activatory ions. Moreover, particular members of this class differ in their optimum pH value and substrate specificity. These results shed new light on the widely accepted classification of plant nucleases which is based on the assumption that the catalytic requirements of plant nucleases reflect their phylogenetic origin. Our results imply the need to redefine the understanding of the term 'nuclease I'. Analysis of the phylogenetic relationships between S1-like enzymes shows that plant representatives of this family evolve toward an increase in catalytic diversity. The importance of this process for the biological functions of plant S1-type enzymes is discussed.

Variations in the enzymatic activity of S1-type nucleases results from differences in their active site structures.

BACKGROUND: S1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn2+-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non­zinc-dependent S1-like nucleases and what is the molecular basis of their activities. METHODS: S1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay. RESULTS: We discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues. CONCLUSIONS: Our findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases. GENERAL SIGNIFICANCE: Our findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non­zinc-dependent members of this family.

Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases.

BACKGROUND: The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. RESULTS: Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. CONCLUSIONS: In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

Histone-lysine N-methyltransferase 2 (KMT2) complexes - a new perspective.

Histone H3 Lys4 (H3K4) methylation is catalyzed by the Histone-Lysine N-Methyltransferase 2 (KMT2) protein family, and its members are required for gene expression control. In vertebrates, the KMT2s function in large multisubunit complexes known as COMPASS or COMPASS-like complexes (COMplex of Proteins ASsociated with Set1). The activity of these complexes is critical for proper development, and mutation-induced defects in their functioning have frequently been found in human cancers. Moreover, inherited or de novo mutations in KMT2 genes are among the etiological factors in neurodevelopmental disorders such as Kabuki and Kleefstra syndromes. The canonical role of KMT2s is to catalyze H3K4 methylation, which results in a permissive chromatin environment that drives gene expression. However, current findings described in this review demonstrate that these enzymes can regulate processes that are not dependent on methylation: noncatalytic functions of KMT2s include DNA damage response, cell division, and metabolic activities. Moreover, these enzymes may also methylate non-histone substrates and play a methylation-dependent function in the DNA damage response. In this review, we present an overview of the new, noncanonical activities of KMT2 complexes in a variety of cellular processes. These discoveries may have crucial implications for understanding the functions of these methyltransferases in developmental processes, disease, and epigenome-targeting therapeutic strategies in the future.

Nuclear localization and actions of the insulin-like growth factor 1 (IGF-1) system components: Transcriptional regulation and DNA damage response.

Insulin-like growth factor (IGF) system stimulates growth, proliferation, and regulates differentiation of cells in a tissue-specific manner. It is composed of two insulin-like growth factors (IGF-1 and IGF-2), six insulin-like growth factor-binding proteins (IGFBPs), and two insulin-like growth factor receptors (IGF-1R and IGF-2R). IGF actions take place mostly through the activation of the plasma membrane-bound IGF-Rs by the circulating ligands (IGFs) released from the IGFBPs that stabilize their levels in the serum. This review focuses on the IGF-1 part of the system. The IGF-1 gene, which is expressed mainly in the liver as well as in other tissues, comprises six alternatively spliced exons that code for three protein isoforms (pro-IGF-1A, pro-IGF-1B, and pro-IGF-1C), which are processed to mature IGF-1 and E-peptides. The IGF-1R undergoes autophosphorylation, resulting in a signaling cascade involving numerous cytoplasmic proteins such as AKT and MAPKs, which regulate the expression of target genes. However, a more complex picture of the axis has recently emerged with all its components being translocated to the nuclear compartment. IGF-1R takes part in the regulation of gene expression by forming transcription complexes, modifying the activity of chromatin remodeling proteins, and participating in DNA damage tolerance mechanisms. Four IGFBPs contain a nuclear localization signal (NLS), which targets them to the nucleus, where they regulate gene expression (IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6) and DNA damage repair (IGFBP-3 and IGFBP-6). Last but not least, the IGF-1B isoform has been reported to be localized in the nuclear compartment. However, no specific molecular actions have been assigned to the nuclear pro-IGF-1B or its derivative EB peptide. Therefore, further studies are needed to shed light on their nuclear activity. These recently uncovered nuclear actions of different components of the IGF-1 axis are relevant in cancer cell biology and are discussed in this review.

A novel method for cloning of coding sequences of highly toxic proteins.

BACKGROUND: During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process. METHODS: Working to solve the problem of DNase II-like cDNA cloning, we developed a novel cloning approach. The method is based on separate cloning of the 5' and 3' fragments of target cDNA into a vector in such a way that the short Multiple Cloning Site insertion remaining between both fragments changes the reading frame and prevents translation of mRNA arising as a result of promoter leakage. Subsequently, to get the vector with full, uninterrupted Open Reading Frame, the Multiple Cloning Site insertion is removed by in vitro restriction/ligation reactions, utilizing the unique restriction site present in native cDNA. RESULTS: Using this designed method, we cloned a coding sequence of AcDNase II that is extremely toxic for bacteria cells. Then, we demonstrated the usefulness of the construct prepared in this way for overexpression of AcDNase II in eukaryotic cells. CONCLUSIONS: The designed method allows cloning of toxic protein coding sequences that cannot be cloned by standard methods. GENERAL SIGNIFICANCE: Cloning of cDNAs encoding toxic proteins is still a troublesome problem that hinders the progress of numerous studies. The method described here is a convenient solution to cloning problems that are common in research on toxic proteins.

PKA-binding domain of AKAP8 is essential for direct interaction with DPY30 protein.

The main function of the A kinase-anchoring proteins (AKAPs) is to target the cyclic AMP-dependent protein kinase A (PKA) to its cellular substrates through the interaction with its regulatory subunits. Besides anchoring of PKA, AKAP8 participates in regulating the histone H3 lysine 4 (H3K4) histone methyltransferase (HMT) complexes. It is also involved in DNA replication, apoptosis, transcriptional silencing of rRNA genes, alternative splicing, and chromatin condensation during mitosis. In this study, we focused on the interaction between AKAP8 and the core subunit of all known H3K4 HMT complexes-DPY30 protein. Here, we demonstrate that the PKA-binding domain of AKAP8 and the C-terminal domain of DPY30, also called Dpy-30 motif, are crucial for the interaction between these proteins. We show that a single amino acid substitution in DPY30 L69D affects its dimerization and completely abolishes its interaction with AKAP8 and another DPY30-binding partner brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1), which is also AKAP domain-containing protein. We further demonstrate that AKAP8 interacts with DPY30 and the RII alpha regulatory subunit of PKA both in the interphase and in mitotic cells, and we show evidences that AKAP8L, a homologue of AKAP8, interacts with core subunits of the H3K4 HMT complexes, which suggests its role as a potential regulator of these complexes. The results presented here reinforce the analogy between AKAP8-RII alpha and AKAP8-DPY30 interactions, postulated before, and improve our understanding of the complexity of the cellular functions of the AKAP8 protein.

Human papillomaviruses in epigenetic regulations.

Human Papillomaviruses (HPVs) are double-stranded DNA viruses, that infect epithelial cells and are etiologically involved in the development of human cancer. Today, over 200 types of human papillomaviruses are known. They are divided into low-risk and high-risk HPVs depending on their potential to induce carcinogenesis, driven by two major viral oncoproteins, E6 and E7. By interacting with cellular partners, these proteins are involved in interdependent viral and cell cycles in stratified differentiating epithelium, and concomitantly induce epigenetic changes in infected cells and those undergoing malignant transformation. E6 and E7 oncoproteins interact with and/or modulate expression of many proteins involved in epigenetic regulation, including DNA methyltransferases, histone-modifying enzymes and subunits of chromatin remodeling complexes, thereby influencing host cell transcription program. Furthermore, HPV oncoproteins modulate expression of cellular micro RNAs. Most of these epigenetic actions in a complex dynamic interplay participate in the maintenance of persistent infection, cell transformation, and development of invasive cancer by a considerable deregulation of tumor suppressor and oncogenes. In this study, we have undertaken to discuss a number of studies concerning epigenetic regulations in HPV-dependent cells and to focus on those that have biological relevance to cancer progression.

PARP-10, a novel Myc-interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation.

The proto-oncoprotein c-Myc functions as a transcriptional regulator that controls different aspects of cell behavior, including proliferation, differentiation, and apoptosis. In addition, Myc proteins have the potential to transform cells and are deregulated in the majority of human cancers. Several Myc-interacting factors have been described that mediate part of Myc's functions in the control of cell behavior. Here, we describe the isolation of a novel 150 kDa protein, designated PARP-10, that interacts with Myc. PARP-10 possesses domains with homology to RNA recognition motifs and to poly(ADP-ribose) polymerases (PARP). Molecular modeling and biochemical analysis define a PARP domain that is capable of ADP-ribosylating PARP-10 itself and core histones, but neither Myc nor Max. PARP-10 is localized to the nuclear and cytoplasmic compartments that is controlled at least in part by a Leu-rich nuclear export sequence (NES). Functionally, PARP-10 inhibits c-Myc- and E1A-mediated cotransformation of rat embryo fibroblasts, a function that is independent of PARP activity but that depends on a functional NES. Together, our findings define a novel PARP enzyme involved in the control of cell proliferation.

The evaluation of ghrelin mRNA expression in human somatotroph adenomas.

Ghrelin is one of the peptides involved into GH-release, binding to specific GHS receptors on hypothalamus and pituitary. The ghrelin peptide and ghrelin mRNA have been detected in several regions of hypothalamus, in normal pituitary, as well as in various types of pituitary adenoma, with different levels of expression in different tumour types. We decided to determine the expression of ghrelin in somatotroph adenomas. Human pituitary somatotroph adenoma tissues were obtained at the time of transsphenoidal surgery from 3 acromegalic patients and studied for ghrelin mRNA expression. Before surgery each patient received a somatostatin analogue treatment at doses 20 mg, 30 mg, 30 mg at 30 days intervals. 20 mg of each tissue sample was used for the isolation of total cellular RNA. The reverse transcription and real-time PCR were performed according to Korbonits et al. method. The reverse transcription of total RNA to cDNA was performed using Super Script TM Rnase H RT kit according to manufacturer protocol. We wished to determine the number of copies of ghrelin gene within the single cell. We used the beta-actin, and the GAPDH genes as a reference molecules for standard curve calculation. Ghrelin mRNA was not detected in any examined tissues. We postulate that the absence of the ghrelin gene transcript is mainly due to the treatment with somatostatin analogues administered preoperatively, which could have suppressed the ghrelin gene transcription.

[Diagnosis of papilloma viruses in cervical cancer in 414 women from Wielkopolska region by the immunohistochemical assessment]. / Identyfikacja wirusów brodawczaka w 414 przypadkach raka inwazyjnego szyjki macicy u kobiet regionu wielkopolskiego w powiazaniu z badaniami immunohistochemicznymi.

OBJECTIVES: Cervical cancer in Poland is second most common type of cancer, after breast cancer. There are known risk factors of cervical cancer development and the most serious one is human papilloma virus infection (HPV). DESIGN: The aim of our paper is present the result study 414 cervical cancer of women from Wielkopolska region treated at the Department of Gynecology and Obstetrics, Karol Marcinkowski University of Medical Sciences in Poznan. MATERIALS AND METHODS: In morphological study grading, staging and vascular invasion were estimated. In cervical cancer tissue papillomaviruses PCR method were used. In immunohistochemical study, expression of proteins checking the cell cycle, share in signal transduction to nucleus, cell receptors for steroid hormones and viruses oncogenic proteins were investigated. In the part of cancer gene mutation of p53 (60 cancers) i k-RAS (40 cancers) were searched. RESULTS: In cancers HPV 16/18 infected vascular invasion were more frequently (p < 0.013). No statistically significant difference in cellular proteins expression in the HPV16/18 positive cancers, HPV16/18 negative and cancers without HPV was observed. However significant difference were demonstrated in proteins expression depending from degree of cancer stage. CONCLUSIONS: The result of these studies suggest that super expression for EGFr is poor prognostic factor in the early stage of cancers (I-II0).

[Analysis of cloning rearrangement of T-cell receptor gamma gene in primary cutaneous lymphoma]. / Analiza klonalnych rearanzacji genu gamma receptora limfocytów T w pierwotnych chloniakach skóry T-komórkowych.

The aim of the study was to evaluate the usefulness of the T-cell receptor (TCR) gamma gene rearrangement analysis in the diagnosis of mycosis fungoides (MF) and Sezary syndrome (SS). The analysis of TCR gamma gene rearrangements was performed in patients with MF/SS in different stages and in subjects with inflammatory dermatoses as the control group, using the method of polymerase chain reaction with subsequent separation of products by temperature gradient gel electrophoresis. Dominant clones with TCR-gamma rearrangement were detected in 86.5% of MF/SS skin biopsies and in 67.5% of MF/SS peripheral blood cells whereas in control group in 12% and 15% respectively. Statistically significant differences were found in the occurrence of clonal T-cells in skin infiltrates between patients with MF/SS and control group. Statistical analysis of TCR-gamma rearrangement in peripheral blood cells did not revealed any differences only in patients with early stage (IA) of MF when compared with inflammatory dermatoses. Detection of T-cell receptor gamma gene rearrangement is a valid supplement to histopathological and immunohistochemical examination in cases suspected of MF/SS however the diagnosis should always be based on the analysis of examinations and clinical stage of patients.

Epigenetic mechanisms in virus-induced tumorigenesis.

About 15-20% of human cancers worldwide have viral etiology. Emerging data clearly indicate that several human DNA and RNA viruses, such as human papillomavirus, Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, hepatitis B virus, hepatitis C virus, and human T-cell lymphotropic virus, contribute to cancer development. Human tumor-associated viruses have evolved multiple molecular mechanisms to disrupt specific cellular pathways to facilitate aberrant replication. Although oncogenic viruses belong to different families, their strategies in human cancer development show many similarities and involve viral-encoded oncoproteins targeting the key cellular proteins that regulate cell growth. Recent studies show that virus and host interactions also occur at the epigenetic level. In this review, we summarize the published information related to the interactions between viral proteins and epigenetic machinery which lead to alterations in the epigenetic landscape of the cell contributing to carcinogenesis.

Histone modifiers in cancer: friends or foes?

Covalent modifications of histones can regulate all DNA-dependent processes. In the last few years, it has become more and more evident that histone modifications are key players in the regulation of chromatin states and dynamics as well as in gene expression. Therefore, histone modifications and the enzymatic machineries that set them are crucial regulators that can control cellular proliferation, differentiation, plasticity, and malignancy processes. This review discusses the biology and biochemistry of covalent histone posttranslational modifications (PTMs) and evaluates the dual role of their modifiers in cancer: as oncogenes that can initiate and amplify tumorigenesis or as tumor suppressors.

Characterization of nucleases involved in seedling development of cauliflower.

The ability of cells to control the degradation of their own DNA is a common feature of most living organisms. In plants, extensive hydrolysis of nuclear DNA occurs during different forms of programmed cell death (PCD). In addition to the removal of unwanted cells, the PCD process allows for the remobilization of cellular constituents, including the products of DNA hydrolysis. Although programmed cell death occurs widely during normal development and plant defense responses to pathogens, only one class of deoxyribonucleases, the S1 type, involved in these processes, has been well characterized. Using DNA-SDS-PAGE, we identified the activities of 14 deoxyribonucleases expressed in different organs of cauliflower seeds, seedlings and the flower head. These enzymes represent several classes based on their substrate specificity and ion dependency. In addition to four Zn(2+)-dependent enzymes, we identified five Ca(2+)-dependent, two Mg(2+)-dependent, three Ca(2+)/Mg(2+)-dependent and one nuclease whose activities seem to be independent of any divalent cations. We also identified a set of DNases whose expression seems to be common for different organs and different stages of development, as well as a few highly tissue-specific nucleases. Expression of three nucleases was inducible by drought stress and hydrogen peroxide.

Identification of a specific IgE-binding protein from narrow-leafed lupin (L. Angustifolius) seeds.

UNLABELLED: Since lupin has been introduced as a food ingredient on the market there are more and more reports concerning its allergenic properties. However, only few narrow-leafed lupin proteins have yet been characterized as specific IgE-binding molecules and identified. The aim of the study has been to find and identify the main narrow-leafed lupin globulins that bind to specific IgEs from the sera of lupin-allergic people. Isolated lupin globulins were subjected to immunoblotting with the sera from people who suffered from lupin allergy. Incubation with alpha-methyl-D-galactopyranoside was performed to eliminate possible binding of unspecific human IgEs. The proteins binding specific IgEs from lupin-allergic patients' sera were identified by means of mass spectrometry. Western blot analysis revealed 2 signals corresponding to lupin globulins that bound to specific IgEs from the sera of people allergic to lupin. The globulins were identified as conglutin-gamma and its smaller subunit. The results suggested that individuals that displayed lupin allergy symptoms reacted to conglutin-gamma. PRACTICAL APPLICATION: The results of the study can contribute to identification of yet undetected allergens of narrow-leafed lupin. This, in turn, can make lupin-fortified products safer for the consumers.

Overlap of the gene encoding the novel poly(ADP-ribose) polymerase Parp10 with the plectin 1 gene and common use of exon sequences.

We have recently identified PARP10 as a novel functional poly(ADP-ribose) polymerase. The gene encoding PARP10 is conserved in vertebrates but no orthologs were found in lower organisms. In addition to the poly(ADP-ribose) polymerase domain, PARP10 possesses several additional sequence motifs, including an RNA recognition motif and two ubiquitin interaction motifs. We characterized the murine genomic locus of the Parp10 gene. We noticed that 3' Parp10 sequences overlapped with the plectin 1 gene in a head-to-tail arrangement. Detailed analyses revealed that the two most 3' Parp10 exons (exons 10 and 11) are also used for plectin 1. While these two exons code for part of the poly(ADP-ribose) polymerase domain in Parp10, they are noncoding for plectin 1 due to the lack of appropriate start codons. Furthermore our findings suggest that at least one of the plectin 1 promoters is located within intron 9 of the Parp10 gene.