Number Of Nonhistone Proteins Research Articles

SMYD2 targets RIPK1 and restricts TNF-induced apoptosis and necroptosis to support colon tumor growth

SMYD2 is a histone methyltransferase, which methylates both histone H3K4 as well as a number of non-histone proteins. Dysregulation of SMYD2 has been associated with several diseases including cancer. In the present study, we investigated whether and how SMYD2 might contribute to colorectal cancer. Increased expression levels of SMYD2 were detected in human and murine colon tumor tissues compared to tumor-free tissues. SMYD2 deficiency in colonic tumor cells strongly decreased tumor growth in two independent experimental cancer models. On a molecular level, SMYD2 deficiency sensitized colonic tumor cells to TNF-induced apoptosis and necroptosis without affecting cell proliferation. Moreover, we found that SMYD2 targeted RIPK1 and inhibited the phosphorylation of RIPK1. Finally, in a translational approach, pharmacological inhibition of SMYD2 attenuated colonic tumor growth. Collectively, our data show that SMYD2 is crucial for colon tumor growth and inhibits TNF-induced apoptosis and necroptosis.

Protein Crotonylation Expert Review: A New Lens to Take Post-Translational Modifications and Cell Biology to New Heights.

Genome regulation, temporal and spatial variations in cell function, continues to puzzle and interest life scientists who aim to unravel the molecular basis of human health and disease, not to mention plant biology and ecosystem diversity. Despite important advances in epigenomics and protein post-translational modifications over the past decade, there is a need for new conceptual lenses to understand biological mechanisms that can help unravel the fundamental regulatory questions in genomes and the cell. To these ends, lys crotonylation (Kcr) is a reversible protein modification catalyzed by protein crotonyl transferases and decrotonylases. First identified on histones, Kcr regulates cellular processes at the chromatin level. Research thus far has revealed that Kcr marks promoter sites of active genes and potential enhancers. Eventually, Kcr on a number of nonhistone proteins was reported. The abundance of Kcr on ribosomal and myofilament proteins indicates its functional roles in protein synthesis and muscle contraction. Kcr has also been associated with pluripotency, spermiogenesis, and DNA repair. In plants, large-scale mass spectrometry-based experiments validated the roles of Kcr in photosynthesis. In this expert review, we present the latest thinking and findings on lys crotonylation with an eye to regulation of cell biology. We discuss the enrichment techniques, putative biological functions, and challenges associated with studying this protein modification with vast biological implications. Finally, we reflect on the future outlook about the broader relevance of Kcr in animals, microbes, and plant species.

The Clinical Significance of SIRT2 in Malignancies: A Tumor Suppressor or an Oncogene?

Sirtuin 2 (SIRT2) is a member of the sirtuin protein family. It is a Class III histone deacetylase (HDACs) and predominantly localized to the cytosol. SIRT2 deacetylates histones and a number of non-histone proteins and plays a pivotal role in various physiologic processes. Previously, SIRT2 has been considered indispensable during carcinogenesis; however, there is now a significant controversy regarding whether SIRT2 is an oncogene or a tumor suppressor. The purpose of this review is to summarize the physiological functions of SIRT2 and its mechanisms in cancer. We will focus on five malignancies (breast cancer, non-small cell lung cancer, hepatocellular carcinoma, colorectal cancer, and glioma) to describe the current status of SIRT2 research and discuss the clinical evaluation of SIRT2 expression and the use of SIRT2 inhibitors.

Abstract A061: Targeting PRMT5 enhances the response to CDK4/6 inhibitors in multiple cancer types

Abstract CDK4 is a central regulator of cell cycle progression and is frequently hyper-activated by various genomic changes in numerous cancer types. CDK4/6 inhibitors such as palbociclib are approved for treatment of HR-positive breast cancer and has shown efficacy across many cancers. However, CDK4/6 inhibitors are best used in combination therapy rather than as a monotherapy. Finding novel combinations that can enhance the efficacy of CDK4/6 inhibitors and overcome or delay the emergence of resistance may provide new treatment strategies for patients.PRMT5 is an emerging target for the treatment of cancer, with the newly developed inhibitor GSK3326595 currently in phase 1/2 clinical trials.PRMT5 is a type II protein methyl-transferase that primarily catalyses the formation of symmetric di-methylarginine modifications. PRMT5 methylates the arginine residue on Histones 2A, 3 and 4 and this is considered an important epigenetic regulatory mechanism. PRMT5 can also methylate a number of non-histone proteins that govern important cellular processes including, cell signalling, RNA nucleo-cytoplasmic transport, pre-RNA processing, and ribosome biogenesis. The combined inhibition of CDK4 and PRMT5 can be seen as a promising strategy for the treatment of many cancers. A high throughput drug screen with a boutique library of targeted therapeutics was performed in order to identify potential targets whose inhibition enhances palbociclib efficacy. The effectiveness of combination treatment was evaluated by measuring cell proliferation clonogenicity, and cell death. To understand the mechanism/s of response to CDK4 and PRMT5 inhibitors, we utilized RPPA and RNA sequencing assays. In addition to this, western blotting, PCR and real-time PCR were also used to study both gene and protein expression levels as well as to validate specific targets. Furthermore, gene depletion via shRNA was performed to validate the response to these treatments. Dual inhibition of CDK4/6 and PRMT5 resulted in a robust and more durable response than the single agents in breast cancer, oesophageal, pancreatic and melanoma cell lines. The response to this combination was observed in both p53-wild type and mutant cell lines. High throughput proteomic and genomic analysis revealed that Inhibition of CDK4/6 and/or PRMT5 acts in p53-dependent and independent mechanisms. In p53 wild type, the combination of palbociclib and GSKGSK3326595 resulted in a permenant state of cell cycle arrest, whereas in p53-mutant cells the combinaiton caused mitotic catastrophe and induced cell death. In conclusion, Inhibition of PRMT5 enhances the response to CDK4/6 inhibitors and the combination of palbociclib and GSK3326595 is a potentially promising treatment strategy for various cancers. Citation Format: Shatha AbuHammad, Grant A McArthur, Karen Sheppard. Targeting PRMT5 enhances the response to CDK4/6 inhibitors in multiple cancer types [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A061. doi:10.1158/1535-7163.TARG-19-A061

Ultradeep Lysine Crotonylome Reveals the Crotonylation Enhancement on Both Histones and Nonhistone Proteins by SAHA Treatment.

Lysine crotonylation is a newly discovered protein post-translational modification and was reported to share transferases and deacylases with lysine acetylation. The acetyltransferase p300 was reported to also contain crotonyltransferase activity, and class I histone deacetylases were demonstrated to be the major histone decrotonylases. However, the decrotonylases for nonhistone proteins are unclear. Moreover, because of the lack of high-quality pan-antibodies, large-scale analysis of crotonylome still remains a challenge. In this work, we comprehensively studied lysine crotonylome on both histones and nonhistone proteins upon SAHA treatment and dramatically identified 10 163 lysine crotonylation sites in A549 cells. This is the first identification of tens of thousands of lysine crotonylation sites and also the largest lysine crotonylome data set up to now. Moreover, a parallel-reaction-monitoring-based experiment was performed for validation, which presented highly consistent results with the SILAC experiments. By intensive bioinformatic analysis, it was found that lysine crotonylation participates in a wide range of biological functions and processes. More importantly, it was revealed that both the crotonylation and acetylation levels of most core histones sites and a number of nonhistone proteins as well as some known substrates of class IIa and IIb HDACs were up-regulated after SAHA treatment. These results suggest that SAHA may have decrotonylation inhibitory activities on both histones and nonhistone proteins by inhibiting HDACs.

The Role of Histone Deacetylase (HDAC) as a Biomarker in Cancer

Histone acetylases [HAT] and histone deacetylases [HDACs] are responsible for the addition and removal of acetyl-groups to or from specific lysine residues located within histone tails and a number of non-histone proteins. HDACs, as one of the epigenetic mechanisms, play a central role in the regulation of cellular properties that related to development and progression of cancer. Recently, researches began to focus on the expression patterns of HDAC isoforms as a biomarker in cancer. It could be used to find new agents that are very effective in inducing apoptosis, differentiation, and/or cell growth arrest in neoplasia. Approximately, all of studies showed HDAC expression level differ in human tumors. For example, in most tumor entities class I HDAC expression was higher in late stage, high-grade tumors with strong proliferative activity and Class II HDACs down regulated in human tumors and high expression in some tumors was linked to a better prognosis. Thus, this factor allowed to opens new possibilities for a molecularly targeted approach to treatment.

Abstract 2953: Involvement of HDAC11 in cell division cycle

Abstract Histone deacetylases (HDACs) are a group of enzymes that catalyze removal of acetyl groups from a ε-N-acetyl lysine amino acid on histones. A number of non-histone proteins are also known to be deacetylated by these enzymes. Hence, these proteins are now also being referred to as lysine deacetylases (KDAC). Based on function and DNA sequence similarity, HDAC proteins are classified in four groups; Class I, Class II, Class III and Class IV HDACs. HDAC11, the only member of Class IV HDAC, shows a DNA sequence similarity with both Class I and Class II HDACs. In general, HDAC proteins are known as transcriptional inhibitors and are involved in cellular growth, development and apoptosis. Unlike other HDACs, function of HDAC11 is not widely studied. Although this enzyme is reported as a transcriptional inhibitor, little is known about its role in connection to cell division cycle or cell density. Analysis of our microarray data shows that Balb/C 3T3 cells, stimulated with PDGF and plasma, contain considerably reduced level of HDAC11 transcript compared to that in the quiescent Balb/C 3T3 cells. The protein level under similar conditions mimics the pattern of transcript level. These results indicate that as cells leave quiescence state HDAC11 expression is down-regulated or may be reduction of HDAC11 expression causes quiescent cells to enter cell cycle. It may also be possible that cell-cell contact inhibition positively regulates HDAC11 expression and as the cell grows in presence of PDGF and plasma, the contact inhibitory property is lost which in turn causes the reduction of the protein expression. Further work is in progress to elucidate the role of HDAC11 with respect to cell cycle regulation or cell density. 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 2953. doi:10.1158/1538-7445.AM2011-2953

Enhancement of Nuclear Factor-κB Acetylation by Coactivator p300 and HIV-1 Tat Proteins

Nuclear factor (NF)-kappaB transcription factors are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. Transcription of the human immunodeficiency virus type 1 (HIV-1) genome depends on the intracellular environment where the integrate viral DNA is regulated by a complex interplay among viral regulatory proteins, such as Tat, and host cellular transcription factors, such as NF-kappaB, interacting with the viral long terminal repeat region. CBP (CREB-binding protein) and p300, containing an intrinsic histone acetyltransferase (HAT) activity, have emerged as coactivators for various DNA-binding transcription factors. Here, we show that the p50 subunit as well as the p50/p65 of NF-kappaB, and not other factors such as SP1, TFIIB, polymerase II, TFIIA, or p65, can be acetylated by CBP/p300 HAT domain. Acetylation of p50 was completely dependent on the presence of both HAT domain and Tat proteins, implying that Tat influences the transcription machinery by aiding CBP/p300 to acquire new partners and increase its functional repertoire. Three lysines, Lys-431, Lys-440, and Lys-441 in p50 were all acetylated in vitro, and a sequence similarity among p50, p53, Tat, and activin receptor type I on these particular lysines was observed. All proteins have been shown to be acetylated by the CBP/p300 HAT domain. Acetylated p50 increases its DNA binding properties, as evident by streptavidin/biotin pull-down assays when using labeled NF-kappaB oligonucleotides. Increased DNA binding on HIV-1 long terminal repeat coincided with increases in the rate of transcription. Therefore, we propose that acetylation of the DNA binding domain of NF-kappaB aids in nuclear translocation and enhanced transcription and also suggest that the substrate specificity of CBP/p300 can be altered by small peptide molecules, such as HIV-encoded Tat.

Mass isolation of polytene nuclei of Tokunagayusurika akamushi (Diptera, Chironomidae): Biochemical and morphological characterization.

A method for the mass isolation of polytene nuclei of T. akamushi is described. Isolated nuclei were studied with light and electron microscopy, SDS-gel electrophoresis and DNA fragment analysis after micrococcal nuclease digestion. Histones were the major components of the chromosomal proteins. A number of non-histone proteins with a wide range of molecular weights were present, but their quantities were relatively low. The sizes of the DNA fragments cleaved by micrococcal nuclease suggested the presence of nucleosomal structures in the polytene chromosomes. The size of the DNA repeat was about 200 base pairs.

A reciprocal relationship between contents of free ubiquitin and protein A24, its conjugate with histone 2A, in chromatin fractions obtained by the DNase II, Mg ++ procedure

Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage ( Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA 74 , 864–868 ). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H 2SO 4 soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH 2-terminal amino acid and its amino acid composition.

Chromatin structure visualization by immunoelectron microscopy

Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 Å, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 Å. This size is compatible with a model where one layer of gamma globulin molecules 110 Å long encircles a sphere of chromatin 100 Å in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting that most of the chromatin beads contain this type of histone.

Nuclear proteins: I. Electrophoretic comparison of mouse nucleoli, heterochromatin, euchromatin and contractile proteins

Summary A number of observations suggest there are some non-histone protein unique to constitutive heterochromatin which are responsible for some of its properties such as interphase condensation, C-banding, frequent decreased quinacrine fluorescence, ectopic pairing, and ability to cause position effect. To investigate this, mouse nuclei were disrupted by sonication and separated by differential centrifugation into heterochromatin+nucleoli, heterochromatin, intermediate, euchromatin pellet and euchromatin supernatant fractions. The non-histone proteins of these fractions were examined by SDS slab gel electrophoresis and compared with the proteins of the cytoplasm, nucleoplasm, cytoplasmic ribosomes, nuclear membrane, and to tubulin, actin, myosin and other muscle proteins. There were a number of non-histone proteins that were unique to or present in greater quantity in the hterochromatin+nucleoli fraction than in the euchromatin. Some of the chromatin non-histone proteins co-migrated with muscle proteins by SDS gel electrophoresis.