Properties and Mechanisms of Deletions, Insertions, and Substitutions in the Evolutionary History of SARS-CoV-2.

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus's macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis.

DNA polymerase ε and δ variants drive mutagenesis in polypurine tracts in human tumors.

Alterations in the exonuclease domain of DNA polymerase ε cause ultramutated cancers. These cancers accumulate AGA>ATA transversions; however, their genomic features beyond the trinucleotide motifs are obscure. We analyze the extended DNA context of ultramutation using whole-exome sequencing data from 524 endometrial and 395 colorectal tumors. We find that G>T transversions in POLE-mutant tumors predominantly affect sequences containing at least six consecutive purines, with a striking preference for certain positions within polypurine tracts. Using this signature, we develop a machine-learning classifier to identify tumors with hitherto unknown POLE drivers and validate two drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike other pathogenic variants, the E978G substitution affects the polymerase domain of Pol ε. We further show that tumors with POLD1 drivers share the extended signature of POLE ultramutation. These findings expand the understanding of ultramutation mechanisms and highlight peculiar mutagenic properties of polypurine tracts in the human genome.

Non-Random Enrichment of Single-Nucleotide Polymorphisms Associated with Clopidogrel Resistance within Risk Loci Linked to the Severity of Underlying Cardiovascular Diseases: The Role of Admixture.

Cardiovascular disease (CVD) is one of the leading causes of death in Puerto Rico, where clopidogrel is commonly prescribed to prevent ischemic events. Genetic contributors to both a poor clopidogrel response and the severity of CVD have been identified mainly in Europeans. However, the non-random enrichment of single-nucleotide polymorphisms (SNPs) associated with clopidogrel resistance within risk loci linked to underlying CVDs, and the role of admixture, have yet to be tested. This study aimed to assess the possible interaction between genetic biomarkers linked to CVDs and those associated with clopidogrel resistance among admixed Caribbean Hispanics. We identified 50 SNPs significantly associated with CVDs in previous genome-wide association studies (GWASs). These SNPs were combined with another ten SNPs related to clopidogrel resistance in Caribbean Hispanics. We developed Python scripts to determine whether SNPs related to CVDs are in close proximity to those associated with the clopidogrel response. The average and individual local ancestry (LAI) within each locus were inferred, and 60 random SNPs with their corresponding LAIs were generated for enrichment estimation purposes. Our results showed no CVD-linked SNPs in close proximity to those associated with the clopidogrel response among Caribbean Hispanics. Consequently, no genetic loci with a dual predictive role for the risk of CVD severity and clopidogrel resistance were found in this population. Native American ancestry was the most enriched within the risk loci linked to CVDs in this population. The non-random enrichment of disease susceptibility loci with drug-response SNPs is a new frontier in Precision Medicine that needs further attention.

Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells.

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 µM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.

The 29-nucleotide deletion in SARS-CoV: truncated versions of ORF8 are under purifying selection.

BACKGROUND: Accessory proteins have diverse roles in coronavirus pathobiology. One of them in SARS-CoV (the causative agent of the severe acute respiratory syndrome outbreak in 2002-2003) is encoded by the open reading frame 8 (ORF8). Among the most dramatic genomic changes observed in SARS-CoV isolated from patients during the peak of the pandemic in 2003 was the acquisition of a characteristic 29-nucleotide deletion in ORF8. This deletion cause splitting of ORF8 into two smaller ORFs, namely ORF8a and ORF8b. Functional consequences of this event are not entirely clear. RESULTS: Here, we performed evolutionary analyses of ORF8a and ORF8b genes and documented that in both cases the frequency of synonymous mutations was greater than that of nonsynonymous ones. These results suggest that ORF8a and ORF8b are under purifying selection, thus proteins translated from these ORFs are likely to be functionally important. Comparisons with several other SARS-CoV genes revealed that another accessory gene, ORF7a, has a similar ratio of nonsynonymous to synonymous mutations suggesting that ORF8a, ORF8b, and ORF7a are under similar selection pressure. CONCLUSIONS: Our results for SARS-CoV echo the known excess of deletions in the ORF7a-ORF7b-ORF8 complex of accessory genes in SARS-CoV-2. A high frequency of deletions in this gene complex might reflect recurrent searches in "functional space" of various accessory protein combinations that may eventually produce more advantageous configurations of accessory proteins similar to the fixed deletion in the SARS-CoV ORF8 gene.

Deletions across the SARS-CoV-2 Genome: Molecular Mechanisms and Putative Functional Consequences of Deletions in Accessory Genes.

The analysis of deletions may reveal evolutionary trends and provide new insight into the surprising variability and rapidly spreading capability that SARS-CoV-2 has shown since its emergence. To understand the factors governing genomic stability, it is important to define the molecular mechanisms of deletions in the viral genome. In this work, we performed a statistical analysis of deletions. Specifically, we analyzed correlations between deletions in the SARS-CoV-2 genome and repetitive elements and documented a significant association of deletions with runs of identical (poly-) nucleotides and direct repeats. Our analyses of deletions in the accessory genes of SARS-CoV-2 suggested that there may be a hypervariability in ORF7A and ORF8 that is not associated with repetitive elements. Such recurrent search in a "sequence space" of accessory genes (that might be driven by natural selection) did not yet cause increased viability of the SARS-CoV-2 variants. However, deletions in the accessory genes may ultimately produce new variants that are more successful compared to the viral strains with the conventional architecture of the SARS-CoV-2 accessory genes.

No evidence for widespread positive selection on double substitutions within codons in primates and yeasts.

Nucleotide substitutions in protein-coding genes can be divided into synonymous (S) and non-synonymous (N) ones that alter amino acids (including nonsense mutations causing stop codons). The S substitutions are expected to have little effect on function. The N substitutions almost always are affected by strong purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases can modulate the deleterious effect of single N substitutions and, thus, could be subjected to the positive selection. This effect has been demonstrated for mutations in the serine codons, stop codons and double N substitutions in prokaryotes. In all abovementioned cases, a novel technique was applied that allows elucidating the effects of selection on double substitutions considering mutational biases. Here, we applied the same technique to study double N substitutions in eukaryotic lineages of primates and yeast. We identified markedly fewer cases of purifying selection relative to prokaryotes and no evidence of codon double substitutions under positive selection. This is consistent with previous studies of serine codons in primates and yeast. In general, the obtained results strongly suggest that there are major differences between studied pro- and eukaryotes; double substitutions in primates and yeasts largely reflect mutational biases and are not hallmarks of selection. This is especially important in the context of detection of positive selection in codons because it has been suggested that multiple mutations in codons cause false inferences of lineage-specific site positive selection. It is likely that this concern is applicable to previously studied prokaryotes but not to primates and yeasts where markedly fewer double substitutions are affected by positive selection.

Elimination of LRVs Elicits Different Responses in Leishmania spp.

Leishmaniaviruses (LRVs) have been demonstrated to enhance progression of leishmaniasis, a vector-transmitted disease with a wide range of clinical manifestations that is caused by flagellates of the genus Leishmania. Here, we used two previously proposed strategies of the LRV ablation to shed light on the relationships of two Leishmania spp. with their respective viral species (L. guyanensis, LRV1 and L. major, LRV2) and demonstrated considerable difference between two studied systems. LRV1 could be easily eliminated by the expression of exogenous capsids regardless of their origin (the same or distantly related LRV1 strains, or even LRV2), while LRV2 was only partially depleted in the case of the native capsid overexpression. The striking differences were also observed in the effects of complete viral elimination with 2'C-methyladenosine (2-CMA) on the transcriptional profiles of these two Leishmania spp. While virtually no differentially expressed genes were detected after the LRV1 removal from L. guyanensis, the response of L. major after ablation of LRV2 involved 87 genes, the analysis of which suggested a considerable stress experienced even after several passages following the treatment. This effect on L. major was also reflected in a significant decrease of the proliferation rate, not documented in L. guyanensis and naturally virus-free strain of L. major. Our findings suggest that integration of L. major with LRV2 is deeper compared with that of L. guyanensis with LRV1. We presume this determines different effects of the viral presence on the Leishmania spp. infections. IMPORTANCE Leishmania spp. represent human pathogens that cause leishmaniasis, a widespread parasitic disease with mild to fatal clinical manifestations. Some strains of leishmaniae bear leishmaniaviruses (LRVs), and this has been shown to aggravate disease course. We investigated the relationships of two distally related Leishmania spp. with their respective LRVs using different strategies of virus removal. Our results suggest the South American L. guyanensis easily loses its virus with no important consequences for the parasite in the laboratory culture. Conversely, the Old-World L. major is refractory to virus removal and experiences a prominent stress if this removal is nonetheless completed. The drastically different levels of integration between the studied Leishmania spp. and their viruses suggest distinct effects of the viral presence on infections in these species of parasites.

Template switching and duplications in SARS-CoV-2 genomes give rise to insertion variants that merit monitoring.

The appearance of multiple new SARS-CoV-2 variants during the COVID-19 pandemic is a matter of grave concern. Some of these variants, such as B.1.617.2, B.1.1.7, and B.1.351, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the Spike (S) protein is thought to be a determinant of SARS-CoV-2 virulence. Here, we identify 346 unique inserts of different lengths in SARS-CoV-2 genomes and present evidence that these inserts reflect actual virus variance rather than sequencing artifacts. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. At least three inserts in the N-terminal domain of the S protein are predicted to lead to escape from neutralizing antibodies, whereas other inserts might result in escape from T-cell immunity. Thus, inserts in the S protein can affect its antigenic properties and merit monitoring.

Unravelling roles of error-prone DNA polymerases in shaping cancer genomes.

Mutagenesis is a key hallmark and enabling characteristic of cancer cells, yet the diverse underlying mutagenic mechanisms that shape cancer genomes are not understood. This review will consider the emerging challenge of determining how DNA damage response pathways-both tolerance and repair-act upon specific forms of DNA damage to generate mutations characteristic of tumors. DNA polymerases are typically the ultimate mutagenic effectors of DNA repair pathways. Therefore, understanding the contributions of DNA polymerases is critical to develop a more comprehensive picture of mutagenic mechanisms in tumors. Selection of an appropriate DNA polymerase-whether error-free or error-prone-for a particular DNA template is critical to the maintenance of genome stability. We review different modes of DNA polymerase dysregulation including mutation, polymorphism, and over-expression of the polymerases themselves or their associated activators. Based upon recent findings connecting DNA polymerases with specific mechanisms of mutagenesis, we propose that compensation for DNA repair defects by error-prone polymerases may be a general paradigm molding the mutational landscape of cancer cells. Notably, we demonstrate that correlation of error-prone polymerase expression with mutation burden in a subset of patient tumors from The Cancer Genome Atlas can identify mechanistic hypotheses for further testing. We contrast experimental approaches from broad, genome-wide strategies to approaches with a narrower focus on a few hundred base pairs of DNA. In addition, we consider recent developments in computational annotation of patient tumor data to identify patterns of mutagenesis. Finally, we discuss the innovations and future experiments that will develop a more comprehensive portrait of mutagenic mechanisms in human tumors.

The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA.

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681-684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence "wildtype" spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

AID and APOBECs as Multifaceted Intrinsic Virus-Restricting Factors: Emerging Concepts in the Light of COVID-19.

The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.

DNA Methylation, Deamination, and Translesion Synthesis Combine to Generate Footprint Mutations in Cancer Driver Genes in B-Cell Derived Lymphomas and Other Cancers.

Cancer genomes harbor numerous genomic alterations and many cancers accumulate thousands of nucleotide sequence variations. A prominent fraction of these mutations arises as a consequence of the off-target activity of DNA/RNA editing cytosine deaminases followed by the replication/repair of edited sites by DNA polymerases (pol), as deduced from the analysis of the DNA sequence context of mutations in different tumor tissues. We have used the weight matrix (sequence profile) approach to analyze mutagenesis due to Activation Induced Deaminase (AID) and two error-prone DNA polymerases. Control experiments using shuffled weight matrices and somatic mutations in immunoglobulin genes confirmed the power of this method. Analysis of somatic mutations in various cancers suggested that AID and DNA polymerases η and θ contribute to mutagenesis in contexts that almost universally correlate with the context of mutations in A:T and G:C sites during the affinity maturation of immunoglobulin genes. Previously, we demonstrated that AID contributes to mutagenesis in (de)methylated genomic DNA in various cancers. Our current analysis of methylation data from malignant lymphomas suggests that driver genes are subject to different (de)methylation processes than non-driver genes and, in addition to AID, the activity of pols η and θ contributes to the establishment of methylation-dependent mutation profiles. This may reflect the functional importance of interplay between mutagenesis in cancer and (de)methylation processes in different groups of genes. The resulting changes in CpG methylation levels and chromatin modifications are likely to cause changes in the expression levels of driver genes that may affect cancer initiation and/or progression.

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.

Insertions in SARS-CoV-2 genome caused by template switch and duplications give rise to new variants that merit monitoring.

The appearance of multiple new SARS-CoV-2 variants during the winter of 2020-2021 is a matter of grave concern. Some of these new variants, such as B.1.617.2, B.1.1.7, and B.1.351, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the COVID-19 pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on point nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the spike protein is thought to be a determinant of SARS-CoV-2 virulence and other inserts might have contributed to coronavirus pathogenicity as well. Here, we investigate insertions in SARS-CoV-2 genomes and identify 347 unique inserts of different lengths. We present evidence that these inserts reflect actual virus variance rather than sequencing errors. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. We show that inserts in the Spike glycoprotein can affect its antigenic properties and thus merit monitoring. At least, three inserts in the N-terminal domain of the Spike (ins245IME, ins246DSWG, and ins248SSLT) that were first detected in 2021 are predicted to lead to escape from neutralizing antibodies, whereas other inserts might result in escape from T-cell immunity.

Analysis of Stop Codons within Prokaryotic Protein-Coding Genes Suggests Frequent Readthrough Events.

Nonsense mutations turn a coding (sense) codon into an in-frame stop codon that is assumed to result in a truncated protein product. Thus, nonsense substitutions are the hallmark of pseudogenes and are used to identify them. Here we show that in-frame stop codons within bacterial protein-coding genes are widespread. Their evolutionary conservation suggests that many of them are not pseudogenes, since they maintain dN/dS values (ratios of substitution rates at non-synonymous and synonymous sites) significantly lower than 1 (this is a signature of purifying selection in protein-coding regions). We also found that double substitutions in codons-where an intermediate step is a nonsense substitution-show a higher rate of evolution compared to null models, indicating that a stop codon was introduced and then changed back to sense via positive selection. This further supports the notion that nonsense substitutions in bacteria are relatively common and do not necessarily cause pseudogenization. In-frame stop codons may be an important mechanism of regulation: Such codons are likely to cause a substantial decrease of protein expression levels.

Frequent Recombination Events in Leishmania donovani: Mining Population Data.

The Leishmania donovani species complex consists of all L. donovani and L. infantum strains mainly responsible for visceral leishmaniasis (VL). It was suggested that genome rearrangements in Leishmania spp. occur very often, thus enabling parasites to adapt to the different environmental conditions. Some of these rearrangements may be directly linked to the virulence or explain the reduced efficacy of antimonial drugs in some isolates. In the current study, we focused on a large-scale analysis of putative gene conversion events using publicly available datasets. Previous population study of L. donovani suggested that population variability of L. donovani is relatively low, however the authors used masking procedures and strict read selection criteria. We decided to re-analyze DNA-seq data without masking sequences, because we were interested in the most dynamic fraction of the genome. The majority of samples have an excess of putative gene conversion/recombination events in the noncoding regions, however we found an overall excess of putative intrachromosomal gene conversion/recombination in the protein coding genes, compared to putative interchromosomal gene conversion/recombination events.

Palmitoylation of Metazoan Carotenoid Oxygenases.

Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption leads to certain health benefits in humans. Carotenoid cleavage oxygenases (CCOs) are a superfamily of enzymes which oxidatively cleave carotenoids and they are present in all kingdoms of life. Complexity of CCO evolution is high. For example, in this study we serendipitously found a new family of eukaryotic CCOs, the apocarotenoid oxygenase-like (ACOL) family. This family has several members in animal genomes and lacks the animal-specific amino acid motif PDPCK. This motif is likely to be associated with palmitoylation of some animal CCOs. We recently demonstrated that two mammalian members of the carotenoid oxygenase family retinal pigment epithelial-specific 65 kDa protein (RPE65) and beta-carotene oxygenase 2 (BCO2) are palmitoylated proteins. Here we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate ß-carotene. We demonstrate that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that metazoan family members lacking the motif (Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora ACOL) are not palmitoylated. Additionally, we observed that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate ß-carotene. Based on our results we conclude that most metazoan carotenoid oxygenases retain the evolutionarily conserved palmitoylation PDPCK motif to target proteins to internal membranes depending on substrate status. Exceptions are in the secreted BCOL subfamily and the strictly cytosolic ancient ACOL subfamily of carotenoid oxygenases.

Dopamine response gene pathways in dorsal striatum MSNs from a gene expression viewpoint: cAMP-mediated gene networks.

BACKGROUND: Medium spiny neurons (MSNs) comprise the main body (95% in mouse) of the dorsal striatum neurons and represent dopaminoceptive GABAergic neurons. The cAMP (cyclic Adenosine MonoPhosphate)-mediated cascade of excitation and inhibition responses observed in MSN intracellular signal transduction is crucial for neuroscience research due to its involvement in the motor and behavioral functions. In particular, all types of addictions are related to MSNs. Shedding the light on the mechanics of the above-mentioned cascade is of primary importance for this research domain. RESULTS: A mouse model of chronic social conflicts in daily agonistic interactions was used to analyze dorsal striatum neurons genes implicated in cAMP-mediated phosphorylation activation pathways specific for MSNs. Based on expression correlation analysis, we succeeded in dissecting Drd1- and Drd2-dopaminoceptive neurons (D1 and D2, correspondingly) gene pathways. We also found that D1 neurons genes clustering are split into two oppositely correlated states, passive and active ones, the latter apparently corresponding to D1 firing stage upon protein kinase A (PKA) activation. We observed that under defeat stress in chronic social conflicts the loser mice manifest overall depression of dopamine-mediated MSNs activity resulting in previously reported reduced motor activity, while the aggressive mice with positive fighting experience (aggressive mice) feature an increase in both D1-active phase and D2 MSNs genes expression leading to hyperactive behavior pattern corresponded by us before. Based on the alternative transcript isoforms expression analysis, it was assumed that many genes (Drd1, Adora1, Pde10, Ppp1r1b, Gnal), specifically those in D1 neurons, apparently remain transcriptionally repressed via the reversible mechanism of promoter CpG island silencing, resulting in alternative promoter usage following profound reduction in their expression rate. CONCLUSION: Based on the animal stress model dorsal striatum pooled tissue RNA-Seq data restricted to cAMP related genes subset we elucidated MSNs steady states exhaustive projection for the first time. We correspond the existence of D1 active state not explicitly outlined before, and connected with dynamic dopamine neurotransmission cycles. Consequently, we were also able to indicate an oscillated postsynaptic dopamine vs glutamate action pattern in the course of the neurotransmission cycles.