Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

Adenosine A(2A) receptor (A(2A)R)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A(2A)R targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A(2A)R and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A(2A)R-interacting partners have in macrophages. Here, we aimed to identify A(2A)R-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A(2A)R as the bait and a macrophage expression library as the prey. We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A(2A)R-CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPM) cells. We also demonstrated that the A(2A)R is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A(2A)R in macrophages. Conversely, we demonstrate that A(2A)R activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A(2A)R-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes

The increase in cell death rates in caloric restricted cells of the yeast helicase mutant rrm3 is Sir complex dependent

Abstract Calorie restriction (CR), which is a reduction in calorie intake without malnutrition, usually extends lifespan and improves tissue integrity. This report focuses on the relationship between nuclear genomic instability and dietary-restriction and its effect on cell survival. We demonstrate that the cell survival rates of the genomic instability yeast mutant rrm3 change under metabolic restricted conditions. Rrm3 is a DNA helicase, chromosomal replication slows (and potentially stalls) in its absence with increased rates at over 1400 natural pause sites including sites within ribosomal DNA and tRNA genes. Whereas rrm3 mutant cells have lower cell death rates compared to wild type (WT) in growth medium containing normal glucose levels (i.e., 2%), under CR growth conditions cell death rates increase in the rrm3 mutant to levels, which are higher than WT. The silent-information-regulatory (Sir) protein complex and mitochondrial oxidative stress are required for the increase in cell death rates in the rrm3 mutant when cells are transferred from growth medium containing 2% glucose to CR-medium. The Rad53 checkpoint protein is highly phosphorylated in the rrm3 mutant in response to genomic instability in growth medium containing 2% glucose. Under CR, Rad53 phosphorylation is largely reduced in the rrm3 mutant in a Sir-complex dependent manner. Since CR is an adjuvant treatment during chemotherapy, which may target genomic instability in cancer cells, our studies may gain further insight into how these therapy strategies can be improved

Absence of Non-histone Protein Complexes at Natural Chromosomal Pause Sites Results in Reduced Replication Pausing in Aging Yeast Cells

There is substantial evidence that genomic instability increases during aging. Replication pausing (and stalling) at difficult-to-replicate chromosomal sites may induce genomic instability. Interestingly, in aging yeast cells, we observed reduced replication pausing at various natural replication pause sites (RPSs) in ribosomal DNA (rDNA) and non-rDNA locations (e.g., silent replication origins and tRNA genes). The reduced pausing occurs independent of the DNA helicase Rrm3p, which facilitates replication past these non-histone protein-complex-bound RPSs, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR), which extend life span, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR cells, the RPSs are less occupied by their specific non-histone protein complexes (e.g., the preinitiation complex TFIIIC), likely because members of these complexes have primarily cytosolic localization. These conditions may lead to reduced replication pausing and may lower replication stress at these sites during aging

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage

Saccharomyces Rrm3p, a 5′ to 3′ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA

In wild-type Saccharomyces cerevisiae, replication forks slowed during their passage through telomeric C(1–3)A/TG(1–3) tracts. This slowing was greatly exacerbated in the absence of RRM3, shown here to encode a 5′ to 3′ DNA helicase. Rrm3p-dependent fork progression was seen at a modified Chromosome VII-L telomere, at the natural X-bearing Chromosome III-L telomere, and at Y‘-bearing telomeres. Loss of Rrm3p also resulted in replication fork pausing at specific sites in subtelomeric DNA, such as at inactive replication origins, and at internal tracts of C(1–3)A/TG(1–3) DNA. The ATPase/helicase activity of Rrm3p was required for its role in telomeric and subtelomeric DNA replication. Because Rrm3p was telomere-associated in vivo, it likely has a direct role in telomere replication

Bcl-x short-isoform is essential for maintaining homeostasis of multiple tissues

Summary: BCL-2-like protein 1 (BCL2L1) is a key component of cell survival and death mechanisms. Its dysregulation and altered ratio of splicing variants associate with pathologies. However, isoform-specific loss-of-function analysis of BCL2L1 remains unexplored. Here we show the functional impact of genetically inhibiting Bcl-x short-isoform (Bcl-xS) in vivo. Bcl-xS is expressed in most tissues with predominant expression in the spleen and blood cells in mice. Bcl-xS knockout (KO) mice show no overt abnormality until 3 months of age. Thereafter, KO mice develop cardiac hypertrophy with contractile dysfunction and splenomegaly by 6 months. Cardiac fibrosis significantly increases in KO, but the frequency of apoptosis is indistinguishable despite cardiomyopathy. The Akt/mTOR and JNK/cJun signaling are upregulated in male KO heart, and the JNK/cJun is activated with increased Bax expression in KO spleen. These results suggest that Bcl-xS may be dispensable for development but is essential for maintaining the homeostasis of multiple organs

DNA replication fork pausing at the LacO₁₆ repeat after LacI-Rap1 expression.

<p>(A) LacO₁₆-URA3 cells carrying pGAL-LacI-RAP1, pGAL-LacI or the control vector were cultured as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.g004" target="_blank">Fig 4B</a>. CsCl gradient purified DNA was digested with AflII and XbaI and analyzed by two-dimensional gel electrophoresis using the indicated probe. The probe detects a 6.3 kb AflII-XbaI fragment. The LacO₁₆ repeat locates 3.0 kb from the AflII site and 2.9 kb from the XbaI site. RFP represents replication fork pausing. Note that some parts of RFP signal are smearing (ST). The number (%) below each panel denotes the ratio of the signal of RFP to that of total replication intermediates. The arrow indicates the direction of replication fork movement. There is a highly active replication origin 40 kb proximal to the LacO₁₆ repeat insert site (the <i>ADH4</i> locus) on chromosome VII [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.ref082" target="_blank">82</a>]. (B) Effect of <i>mec1Δ</i> mutation on DNA breakage induction. LacO₁₆-URA3 <i>MEC1</i> or <i>mec1Δ</i> cells were transformed with pGAL-LacI (I) or pGAL-LacI-RAP1 (R). Transformants were cultured and DNA was analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.g004" target="_blank">Fig 4B</a>. Cells contain an <i>sml1Δ</i> mutation. Hybridization detects two NcoI-fragments; one from chromosome I (5 kb, marked with INT) and the other from the chromosome VII region (11.6 kb, marked with PRE). DNA breakage at LacO₁₆ generates a 1.3–1.7 kb fragment (marked with CUT) from the 11.6 kb fragment. (C) Effect of <i>mrc1Δ</i> or <i>tof1Δ</i> mutation on DNA breakage induction. LacO₁₆-URA3, LacO₁₆-URA3 <i>mrc1Δ</i> or LacO₁₆-URA3 <i>tof1Δ</i> cells were transformed with pGAL-LacI or pGAL-LacI-RAP1. Transformants were cultured and DNA was analyzed as in (B).</p

DSB induction at the LacO₁₆ repeat after LacI-Rap1 expression.

<p>(A) Schematic of the LacO₁₆ repeat at the <i>ADH4</i> locus on chromosome VII-L. The <i>ADH4</i> locus was marked with a DNA fragment containing the <i>KanMX</i> gene and the <i>URA3</i>^<i>Kl</i> gene. The LacO₁₆ sequence is inserted between the BamHI and EcoRI site. Black bar indicates a hybridization probe for Southern blot. The forward primer (F) and reverse primer (R) were used for a TdT-based DNA end detection assay. Primer F and R are 0.52 kb and 1.1 kb away from the LacO₁₆ insertion site, respectively. (B) Detection of DNA breakage by Southern blot. Wild-type or <i>rad52Δ yku70Δ</i> mutant cells containing the LacO₁₆-URA3 cassette were transformed with pGAL-LacI-RAP1 or pGAL-LacI. Transformants were initially grown in 2% sucrose and then incubated with 2% galactose and 0.5% glucose for 4 hr. Genomic DNA was digested with NcoI and then analyzed by Southern blot using the <i>KanMX</i> gene and a fragment from chromosome I as a probe. Hybridization detects two NcoI-fragments; one from chromosome I (5 kb, marked with INT) and the other from the chromosome VII region (11.6 kb, marked with PRE). DNA breakage at LacO₁₆ generates a 1.3–1.7 kb fragment (marked with CUT) from the 11.6 kb fragment. (C) Detection of DNA ends by TdT-dependent dCTP addition. LacO₁₆-URA3 cells carrying pGAL-LacI-RAP1 or pGAL-LacI were treated as in (B). Genomic DNA was incubated with TdT using dCTP as a substrate and then subjected to PCR using either the forward primer or the reverse primer with a poly(dG)-oligonucleotide. As control, the <i>SMC2</i> locus was amplified by PCR.</p

Requirement of Rap1 on chromosome truncation at TG repeats.

<p>(A) Effect of <i>rap1</i>-degron mutation on colony formation in the presence of CuSO₄. Wild-type or <i>rap1</i>-degron mutant (<i>rap1-(Δ)</i>) cells were serially diluted (10-fold) and spotted on medium containing 0, 0.05 or 0.5 mM CuSO₄. (B) Effect of CuSO₄ concentration on Rap1-(Δ) protein expression. Wild-type or <i>rap1-(Δ)</i> cells were initially grown in the absence of CuSO₄ and then incubated with the indicated concentrations of CuSO₄ for 6 hr. Aliquots of cells were collected and subjected to immunoblotting analysis with anti-Rap1 antibodies. (C) Effect of Rap1 depletion on <i>URA3</i>^<i>Kl</i> marker loss. Wild-type or <i>rap1-(Δ)</i> cells containing the TG₂₅₀ cassette were first maintained in medium selective for <i>URA3</i> and then transferred to non-selective medium containing 0.05 mM CuSO₄. Saturated cultures were diluted and spread on 5-FOA plates to determine the rate of <i>URA3</i>^<i>Kl</i> marker loss. <i>URA3</i>^<i>Kl</i> maker loss was determined as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005283#pgen.1005283.g001" target="_blank">Fig 1C</a>.</p

DSB induction at an artificially elongated telomere.

<p>(A) Schematic of modified VII-L telomeres. Telomeres marked the <i>KanMX</i> marker are generated near the <i>ADH4</i> locus. Triangles and black bars represent TG and lacO sequence, respectively. The VII-L subtelomere sequence is deleted. The black line indicates a hybridization probe. Each chromosome end contains a wild-type length telomere (~300 bp) before LacI-Rap1 expression. Drawing not to scale. (B) Schematic of experimental protocol to detect DNA breaks at an extended telomere. LacI-Rap1 expression converts TG₃₃-LacO₁₆-telomeres to a long telomere mimic. Telomeres become shortened 3–5 bp per generation because of the end-replication problem. Cdc13 binds single-stranded telomeric TG DNA and blocks DNA degradation. Telomere shortening or DSB induction needs to trim off ~300 bp telomeric TG sequence to initiate degradation of the LacO₁₆ repeat. Once the LacO₁₆ repeat is degraded, telomere addition occurs at the TG₃₃ repeat, generating TG-telomere. (C) Effect of LacI-Rap1 expression on the length of LacO₄-telomeres. Cells containing the LacO₄-telomere were transformed with pGAL-LacI-RAP1 or pGAL-LacI. Transformed cells were grown in sucrose to full growth. The culture was then diluted 1000-fold in 2% galctose and 0.5% glucose to allow cells to undergo cell division for 24 hr and aliquots were collected for genomic DNA preparation. This cycle was repeated three times. DNA was digested with HindIII and analyzed by Southern blot using the probe shown in (A). The probe detects the LacO₄-telomere and a 1.8 kb HindIII fragment (Control) from the <i>SMC2</i> locus on chromosome VI. (D) Effect of LacI-Rap1 expression on the length of TG₃₃-LacO₁₆-telomeres. Cells containing the TG₃₃-LacO₁₆-telomere (TG₃₃-LacO₁₆-tel cells) were transformed with pGAL-LacI-RAP1 or pGAL-LacI. Transformants were analyzed as in (C). As control, DNA from cells containing TG-telomere was examined together. The band labeled PRE indicates a fragment containing TG₃₃-LacO₁₆ telomere (~1.2 kb). After DSB induction within or near the LacO₁₆ repeat, this band is converted to a new band (~0.8 kb), which is similar to the fragment containing wild-type length TG-telomeres. The probe also detects a 1.8 kb HindIII fragment (Control) from the <i>SMC2</i> locus on chromosome VI. (E) Effect of telomerase loss on the length of TG₃₃-LacO₁₆-telomeres. TG₃₃-LacO₁₆-tel cells were transformed with pGAL-LacI-RAP1 whereas TG₃₃-LacO₁₆-tel <i>est1Δ</i> cells carrying the <i>URA3</i>-marked <i>EST1</i> plasmid were transformed with pGAL-LacI. Transformants were streaked on plates containing 5-FOA and colonies of Ura^- cells were inoculated in 2% galactose and 0.5% glucose medium and grown to the late log phase (1st dilution). The culture was diluted 1000-fold to allow cells to undergo cell division for 24 hr. This cycle was repeated twice. DNA was analyzed as in (D). TG₃₃-LacO₁₆-tel cells in sucrose (0 dilution) or <i>est1Δ</i> mutants carrying the <i>URA3</i>-marked <i>EST1</i> plasmid (E) in sucrose were examined as control. (F) Effect of r<i>ad52Δ</i> mutation on the length of TG₃₃-LacO₁₆-telomeres after LacI-Rap1 expression. TG₃₃-LacO₁₆-tel wild-type or <i>rad52Δ</i> cells were transformed with pGAL-LacI-RAP1. Transformed cells were analyzed as in (D).</p