Cancer Relevance of Circulating Antibodies Against LINE-1 Antigens in Humans.

Long interspersed nuclear element-1 (LINE-1 or L1), the most abundant family of autonomous retrotransposons occupying over 17% of human DNA, is epigenetically silenced in normal tissues by the mechanisms involving p53 but is frequently derepressed in cancer, suggesting that L1-encoded proteins may act as tumor-associated antigens recognized by the immune system. In this study, we established an immunoassay to detect circulating autoantibodies against L1 proteins in human blood. Using this assay in >2,800 individuals with or without cancer, we observed significantly higher IgG titers against L1-encoded ORF1p and ORF2p in patients with lung, pancreatic, ovarian, esophageal, and liver cancers than in healthy individuals. Remarkably, elevated levels of anti-ORF1p-reactive IgG were observed in patients with cancer with disease stages 1 and 2, indicating that the immune response to L1 antigens can occur in the early phases of carcinogenesis. We concluded that the antibody response against L1 antigens could contribute to the diagnosis and determination of immunoreactivity of tumors among cancer types that frequently escape early detection. SIGNIFICANCE: The discovery of autoantibodies against antigens encoded by L1 retrotransposons in patients with five poorly curable cancer types has potential implications for the detection of an ongoing carcinogenic process and tumor immunoreactivity.

Loss of Astrocytic µ Opioid Receptors Exacerbates Aversion Associated with Morphine Withdrawal in Mice: Role of Mitochondrial Respiration.

Astrocytes express mu/µ opioid receptors, but the function of these receptors remains poorly understood. We evaluated the effects of astrocyte-restricted knockout of µ opioid receptors on reward- and aversion-associated behaviors in mice chronically exposed to morphine. Specifically, one of the floxed alleles of the Oprm1 gene encoding µ opioid receptor 1 was selectively deleted from brain astrocytes in Oprm1 inducible conditional knockout (icKO) mice. These mice did not exhibit changes in locomotor activity, anxiety, or novel object recognition, or in their responses to the acute analgesic effects of morphine. Oprm1 icKO mice displayed increased locomotor activity in response to acute morphine administration but unaltered locomotor sensitization. Oprm1 icKO mice showed normal morphine-induced conditioned place preference but exhibited stronger conditioned place aversion associated with naloxone-precipitated morphine withdrawal. Notably, elevated conditioned place aversion lasted up to 6 weeks in Oprm1 icKO mice. Astrocytes isolated from the brains of Oprm1 icKO mice had unchanged levels of glycolysis but had elevated oxidative phosphorylation. The basal augmentation of oxidative phosphorylation in Oprm1 icKO mice was further exacerbated by naloxone-precipitated withdrawal from morphine and, similar to that for conditioned place aversion, was still present 6 weeks later. Our findings suggest that µ opioid receptors in astrocytes are linked to oxidative phosphorylation and they contribute to long-term changes associated with opioid withdrawal.

Inflammatory response to retrotransposons drives tumor drug resistance that can be prevented by reverse transcriptase inhibitors.

Activation of endogenous retrotransposons frequently occurs in cancer cells and contributes to tumor genomic instability. To test whether inhibition of retrotranspositions has an anticancer effect, we used treatment with the nucleoside reverse transcriptase inhibitor (NRTI) stavudine (STV) in mouse cancer models, MMTV-HER2/Neu and Th-MYCN, that spontaneously develop breast cancer and neuroblastoma, respectively. In both cases, STV in drinking water did not affect tumor incidence nor demonstrate direct antitumor effects. However, STV dramatically extended progression-free survival in both models following an initial complete response to chemotherapy. To approach the mechanism underlying this phenomenon, we analyzed the effect of NRTI on the selection of treatment-resistant variants in tumor cells in culture. Cultivation of mouse breast carcinoma 4T1 in the presence of STV dramatically reduced the frequency of cells capable of surviving treatment with anticancer drugs. Global transcriptome analysis demonstrated that the acquisition of drug resistance by 4T1 cells was accompanied by an increase in the constitutive activity of interferon type I and NF-κB pathways and an elevated expression of LINE-1 elements, which are known to induce inflammatory responses via their products of reverse transcription. Treatment with NRTI reduced NF-κB activity and reverted drug resistance. Furthermore, the inducible expression of LINE-1 stimulated inflammatory response and increased the frequency of drug-resistant variants in a tumor cell population. These results indicate a mechanism by which retrotransposon desilencing can stimulate tumor cell survival during treatment and suggest reverse transcriptase inhibition as a potential therapeutic approach for targeting the development of drug-resistant cancers.

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/ß hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine.

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.

New member of the hormone-sensitive lipase family from the permafrost microbial community.

Siberian permafrost is a unique environment inhabited with diverse groups of microorganisms. Among them, there are numerous producers of biotechnologically relevant enzymes including lipases and esterases. Recently, we have constructed a metagenomic library from a permafrost sample and identified in it several genes coding for potential lipolytic enzymes. In the current work, properties of the recombinant esterases obtained from this library are compared with the previously characterized lipase from Psychrobacter cryohalolentis and other representatives of the hormone-sensitive lipase family.

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

Expression and chaperone-assisted refolding of a new cold-active lipase from Psychrobacter cryohalolentis K5(T).

We describe cloning and expression of genes coding for lipase Lip2Pc and lipase-specific foldase LifPc from a psychrotrophic microorganism Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg (the lense of overcooled brine within permafrost). Upon expression in Escherichiacoli Lip2Pc accumulated in inclusion bodies while chaperone was synthesized in a soluble form. An efficient protocol for solubilization and subsequent refolding of the recombinant lipase in the presence of the truncated chaperone was developed. Using this procedure Lip2Pc with specific activity of 6900U/mg was obtained. Contrary to published data on other lipase-chaperone complexes, refolded Lip2Pc was mostly recovered from the complex with chaperone by metal-affinity chromatography. Recombinant Lip2Pc displayed maximum lipolytic activity at 25°C and pH 8.0 with p-nitrophenyl palmitate (C16) as a substrate. Activity assays conducted at different temperatures revealed that the recombinant Lip2Pc is a cold-adapted lipase with ability to utilize substrates with long (C10-C16) hydrocarbon chains in the temperature range from +5 to +65°C. It demonstrated relatively high stability at temperatures above 60°C and in the presence of various metal ions or organic solvents (ethanol, methanol, etc.). Non-ionic detergents, such as Triton X-100 and Tween 20 decreased Lip2Pc activity and SDS completely inhibited it.

Cloning, purification, and characterization of a cold-adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg.

A psychrotrophic gram-negative bacterium Psychrobacter cryohalolentis K5(T) was previously isolated from a cryopeg within Siberian permafrost and its genome has been completely sequenced. To clone and characterize potential cold-active lipases/esterases produced by P. cryohalolentis K5(T) , we have identified their potential genes by alignment with amino acid sequences of lipases/esterases from related bacteria. One of the targets, EstPc, was cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant protein was produced with a 6x histidine tag at its C-terminus and purified by nickel affinity chromatography. Purified recombinant protein displayed maximum esterolytic activity with p-nitrophenyl butyrate (C4) as a substrate at 35 °C and pH 8.5. Activity assay conducted at different temperatures revealed that EstPc is a cold-adapted esterase which displayed more than 90% of its maximum activity at 0-5 °C. In contrast to many known cold-active enzymes, it possesses relatively high thermostability, preserving more than 60% of activity after incubation for 1 h at 80 °C. It was activated by Ca(2+) , Mn(2+) , and EDTA whereas Zn(+2) , Cu(+2) , Co(+2) , Ni(+2) , and Mg(+2) inhibited it. Various organic solvents (ethanol, methanol and others) inhibited the enzyme. Most non-ionic detergents, such as Triton X-100 and Tween 20 increased the lipase activity while SDS completely inhibited it.