Human AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine in DNA and RNA

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.     

Mapping the in vitro interactome of cardiac sodium (Na+)‐calcium (Ca2+) exchanger 1 (NCX1)

The sodium (Na⁺)‐calcium (Ca²⁺) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca²⁺ homeostasis, serving as the primary mechanism for Ca²⁺ extrusion during relaxation. Dysregulation of NCX1 is observed in end‐stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti‐NCX1 against endogenous NCX1 and (2) anti‐His (where His is histidine) with His‐trigger factor‐NCX1_cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein–protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where “cell communication” and “signal transduction” formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in “cardiovascular disease” which can be explored as novel drug targets in future research.     

Cyclin D1 expression and cell cycle response in DNA mismatch repair-deficient cells upon methylation and UV-C damage

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.     

Progressive sensitivity of trophic levels to warming underlies an elevational gradient in ant–aphid mutualism strength

Although species interactions are often proposed to be stronger at lower latitudes and elevations, few studies have evaluated the mechanisms driving such patterns. In this study, we assessed whether, and by which mechanisms, abiotic changes associated with elevation altered the outcome of an ant–aphid protection mutualism. To do so, we characterized the multi‐trophic interactions among the ant Formica podzolica, the aphid Aphis varians, and aphid natural enemies occurring on the plant Chamerion angustifolium within replicate high and low elevation valleys. Low (versus high) elevation sites had longer summers (snowmelt 13 days earlier) and were on average 1.1°C warmer and 41% drier throughout the year. At low elevations, individual ant colonies consumed approximately double the volume of carbohydrate baits, likely due to a higher foraging tempo, and possibly due to a greater demand for sugar‐ versus protein‐rich resources (as indicated by stable isotope analysis). Wild aphid colonies at low elevations were visited by 1.4‐fold more natural enemies (controlling for variation in aphid abundance), while experimental aphid colonies on potted plants were tended 52% more frequently by ants. As a result, ants increased aphid colony survival by 66% at low elevations but had no detectable effect at high elevations; at low (versus high) elevations aphid colonies without ants had lower survival, demonstrating stronger predator effects, while aphid colonies with ants had higher survival, demonstrating even stronger ant benefits. Analyses for the effects of mean summer temperature yielded qualitatively identical results to those based on elevation. Collectively, these findings support predictions for a greater sensitivity of higher trophic levels to warming and demonstrate how species interactions can vary across environmental gradients due to simultaneous changes in species traits and abundances across multiple trophic levels.     

Lipopolysaccharide and double-stranded RNA up-regulate toll-like receptor 2 independently of myeloid differentiation factor 88

Toll-like receptor 2 (TLR2) is a signaling receptor for a variety of microbial products, including bacterial lipoproteins and peptidoglycan, and is central in initiating immune responses toward Gram-positive bacteria, spirochetes, and mycobacteria. The mechanisms behind regulation of TLR2 protein expression are still not well understood. By using a newly developed monoclonal antibody against mouse TLR2, we detected TLR2 protein expression on macrophages, neutrophils, and dendritic cells. Endogenous macrophage TLR2 localized mostly to the cell membrane, with particular accumulation around phagosomes containing zymosan. Treatment of macrophages with the TLR2 antibody diminished cellular response to lipoproteins and down-regulated membrane TLR2. Marked up-regulation of surface TLR2 was observed on macrophages in response to whole bacteria, lipoproteins, lipopolysaccharide, poly(I-C) (double-stranded RNA), R848, and CpG DNA, and this up-regulation appeared to be a very sensitive marker for the presence of microbial products. Up-regulation of TLR2 in response to stimuli correlated with an increased response to secondary lipoprotein exposure following a low concentration of primary lipoprotein challenge. By comparison, exposure to a larger primary challenge induced a hyporeactive state. Most interestingly, lipopolysaccharide- and double-stranded RNA-induced up-regulation of surface TLR2 in macrophages was found to be MyD88-independent, whereas the up-regulation in response to lipoproteins, R848, and CpG DNA was absent in MyD88-deficient cells. We conclude that complex mechanisms regulate expression and signaling via TLR2. Up-regulation of TLR2 in the presence of low, yet clinically relevant amounts of microbial products may be an important mechanism by which the immune system boosts its response to a beginning infection.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S₅₂₁) were placed in darkness in a buffer containing 50 mM KNO₃, nitrite was excreted from the tissue at rates of 0.08–0.2 mol (g FW)^–1 h^–1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S₅₂₁, although 20–40 mol (g FW)^–1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S₅₂₁ also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S₅₂₁ was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.Abbreviations NR Nitrate reductase - NO Nitric oxide - Ser Serine - WT Wild type