The present invention is directed to secreted and transmembrane polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

Secreted and Transmembrane Polypeptides and Nucleic Acids Encoding the Same

Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

Sumoylation: A Regulatory Protein Modification in Health and Disease

DNA double-strand break (DSB) signalling and repair is crucial to preserve genomic integrity and maintain cellular homeostasis. p53-binding protein 1 (53BP1) is an important regulator of the cellular response to DSBs that promotes the end-joining of distal DNA ends, which is induced during V(D)J and class switch recombination as well as during the fusion of deprotected telomeres. New insights have been gained into the mechanisms underlying the recruitment of 53BP1 to damaged chromatin and how 53BP1 promotes non-homologous end-joining-mediated DSB repair while preventing homologous recombination. From these studies, a model is emerging in which 53BP1 recruitment requires the direct recognition of a DSB-specific histone code and its influence on pathway choice is mediated by mutual antagonism with breast cancer 1 (BRCA1).

Double-strand break repair: 53BP1 comes into focus

Purpose of review Epigenetics investigates heritable changes in gene expression occurring without changes in DNA sequence. Several epigenetic mechanisms, including DNA methylation, histone modifications, and microRNA expression, can change genome function under exogenous influence. Here, we review current evidence indicating that epigenetic alterations mediate toxicity from environmental chemicals. Recent findings In-vitro, animal, and human investigations have identified several classes of environmental chemicals that modify epigenetic marks, including metals (cadmium, arsenic, nickel, chromium, and methylmercury), peroxisome proliferators (trichloroethylene, dichloroacetic acid, and TCA), air pollutants (particulate matter, black carbon, and benzene), and endocrine-disrupting/reproductive toxicants (diethylstilbestrol, bisphenol A, persistent organic pollutants, and dioxin). Most studies conducted so far have been centered on DNA methylation, whereas only a few investigations have studied environmental chemicals in relation to histone modifications and microRNA. Summary For several exposures, it has been proved that chemicals can alter epigenetic marks, and that the same or similar epigenetic alterations can be found in patients with the disease of concern or in diseased tissues. Future prospective investigations are needed to determine whether exposed individuals develop epigenetic alterations over time and, in turn, which such alterations increase the risk of disease. Also, further research is needed to determine whether environmental epigenetic changes are transmitted transgenerationally.

Epigenetics and environmental chemicals.

Chronic exposure to nickel(II), chromium(VI), or inorganic arsenic (iAs) has long been known to increase cancer incidence among affected individuals. Recent epidemiological studies have found that carcinogenic risks associated with chromate and iAs exposures were substantially higher than previously thought, which led to major revisions of the federal standards regulating ambient and drinking water levels. Genotoxic effects of Cr(VI) and iAs are strongly influenced by their intracellular metabolism, which creates several reactive intermediates and byproducts. Toxic metals are capable of potent and surprisingly selective activation of stress-signaling pathways, which are known to contribute to the development of human cancers. Depending on the metal, ascorbate (vitamin C) has been found to act either as a strong enhancer or suppressor of toxic responses in human cells. In addition to genetic damage via both oxidative and nonoxidative (DNA adducts) mechanisms, metals can also cause significant changes in DNA methylation and histone modifications, leading to epigenetic silencing or reactivation of gene expression. In vitro genotoxicity experiments and recent animal carcinogenicity studies provided strong support for the idea that metals can act as cocarcinogens in combination with nonmetal carcinogens. Cocarcinogenic and comutagenic effects of metals are likely to stem from their ability to interfere with DNA repair processes. Overall, metal carcinogenesis appears to require the formation of specific metal complexes, chromosomal damage, and activation of signal transduction pathways promoting survival and expansion of genetically/epigenetically altered cells.

http://pubs.acs.org/doi/pdf/10.1021/tx700198a

Genetic and epigenetic mechanisms in metal carcinogenesis and cocarcinogenesis: nickel, arsenic, and chromium.

Covalent conjugation of the small ubiquitin-like modifier (SUMO) proteins to target proteins regulates many important eukaryotic cellular mechanisms. Although the molecular consequences of the conjugation of SUMO proteins are relatively well understood, little is known about the cellular signals that regulate the modification of their substrates. Here, we show that SUMO-2 and SUMO-3 are required for cells to survive heat shock. Through quantitative labeling techniques, stringent purification of SUMOylated proteins, advanced mass spectrometric technology, and novel techniques of data analysis, we quantified heat shock-induced changes in the SUMOylation state of 766 putative substrates. In response to heat shock, SUMO was polymerized into polySUMO chains and redistributed among a wide range of proteins involved in cell cycle regulation; apoptosis; the trafficking, folding, and degradation of proteins; transcription; translation; and DNA replication, recombination, and repair. This comprehensive proteomic analysis of the substrates of a ubiquitin-like modifier (Ubl) identifies a pervasive role for SUMO proteins in the biologic response to hyperthermic stress.

System-wide changes to SUMO modifications in response to heat shock

Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.

/pdf/sumo-targeted-ubiquitin-e3-ligase-rnf4-is-required-for-the-162163etpq.pdf

SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

Ubc9 is a homologue of the E2 ubiquitin conjugating enzyme and participates in the covalent linking of SUMO-1 molecule to the target protein. In this report we describe a simple and efficient method for obtaining pure human recombinant Ubc9 protein. The purified Ubc9 retained its native structure and was fully active in an in vitro sumoylation assay with the promyelocytic leukaemia (PML) peptide as a substrate. In order to better understand the physiology of Ubc9 protein we examined its levels in several rat tissues. Immunoblot analyses performed on tissue extracts revealed quantitative and qualitative differences in the expression pattern of Ubc9. The Ubc9 protein was present at a high level in spleen and lung. Moderate level of Ubc9 was detected in kidney and liver. Low amount of Ubc9 was observed in brain, whereas the 18 kDa band of Ubc9 was barely visible or absent in heart and skeletal muscle. In heart and muscle extracts the Ubc9 antibodies recognized a 38 kDa protein band. This band was not visible in extracts of other rat tissues. A comparison of the relative levels of Ubc9 mRNA and protein indicated that the overall expression level of Ubc9 was the highest in spleen and lung. In spleen, lung, kidney, brain, liver and heart there was a good correlation between the 18 kDa protein and Ubc9 mRNA levels. In skeletal muscle the Ubc9 mRNA level was unproportionally high comparing to the level of the 18 kDa protein. The presented data indicate that in the rat the expression of the Ubc9 protein appears to have some degree of tissue specificity.

https://ojs.ptbioch.edu.pl/index.php/abp/article/download/3632/2690

Expression level of Ubc9 protein in rat tissues

Nickel, a well-established human carcinogen, was shown to decrease acetylation of histones H4 and H3 in cultured cells. Such a decrease is expected to suppress gene expression. However, nickel is known to not only suppress but also enhance the expression of many genes. So, perhaps, nickel can alter histone acetylation in a more complex way? In a first step of testing this presumption, we examined acetylation status of histones H2A, H2B, H3 and H4, in human (HAE) and rat (NRK) cells exposed to nickel(II) under various conditions. In both cell lines, acetylation of all four histones was down-regulated by nickel(II) in a concentration- and time-dependent manner. Acetylation of histone H2B was suppressed to greater extent than that of the others, with histone H3 being relatively least affected. The analysis of acetylation status of each of the four lysine sites at the N-terminal tail of histone H2B revealed decreases consistent with those observed in the total acetylation patterns, with the K12 and K20 residues being markedly more affected than K5 and K15 residues. Thus, the decrease in acetylation was to some degree site specific. In NRK cells, the observed uniform down-regulation of histone acetylation was consistent with a marked suppression of global gene transcription measured as [3H]-uridine incorporation into mRNA. However, in HAE cells, global RNA expression was transiently increased (in 24 h) before dropping below control after longer exposure (3 days). In conclusion, the effects of Ni(II) on histone acetylation are inhibitory, with their extent depending on the dose and exposure time. This uniform inhibition, however, is not consistently reflected in global RNA expression that in HAE cells may include both increase and decrease of the expression, clearly indicating the involvement of factors other than histone acetylation. The observed effects may contribute to neoplastic transformation of Ni(II)-exposed cells.

/pdf/inhibition-of-core-histones-acetylation-by-carcinogenic-uawpod5ypp.pdf

Inhibition of core histones acetylation by carcinogenic nickel(II).

Molecular mechanisms of nickel-induced carcinogenesis include interactions of Ni(II) cations with histones Previously, we demonstrated in vitro and in cells that Ni(II) cleaved off the -SHHKAKGK C-terminal motif of histone H2A In the present study, Western blotting of histones isolated from rat and human cell lines, cultured for 3-5 days with 005-05 mM Ni(II), revealed time- and dose-dependent appearance of a new band of histone H2B This effect was also induced by Co(II), but not by Cu(II), Cd(II), and Zn(II) Mass spectrometry and amino acid sequencing of proteins from the new band allowed for identification of two derivatives of the major variant of histone H2B The larger protein was histone H2B lacking 16 N-terminal amino acids The smaller one was histone H2B which, in addition to being shortened at the N-terminus, had nine amino acids deleted from its C-terminus At both termini, the truncation occurred between lysine and alanine in the two identical -KAVTK- repeats of histone H2B Also, the truncated H2B proteins had their Q22 residues deamidated and M59 and M62 residues oxidized to sulfoxides, a signature of oxidative stress The truncation did not concur with apoptosis Its mechanism involved activation by Ni(II) treatment of specific nuclear proteolytic enzymes belonging to the calpain family The terminal tails of core histones participate in structuring chromatin and regulating gene expression Therefore, the observed truncation and other modifications of histone H2B may assist in Ni(II) carcinogenesis through epigenetic mechanisms

Filip Golebiowski

Papers

System-wide changes to SUMO modifications in response to heat shock

SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

Expression level of Ubc9 protein in rat tissues

Inhibition of core histones acetylation by carcinogenic nickel(II).

Truncation, deamidation, and oxidation of histone H2B in cells cultured with nickel(II).