ER–mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem‐like cells

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem‐like cells (GSC) being more sensitive to cytotoxic lymphocyte‐mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER–mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER–mitochondria contacts compared to GDCs. Forced ER–mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER–mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.     

rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

Background

Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra.

Results

We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants.

Conclusions

In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7.

     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Development,characterization, validation,and mapping of SSRs derived from <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.<Emphasis Type="Italic">–Moniliophthora perniciosa</Emphasis> interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F₂ Sca 6 × ICS 1 population reference for WBD resistance.     

The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.The S100 proteins comprise a multigene family of low-molecular-weight EF-hand calcium binding and zinc binding proteins (5, 13, 16, 24, 33). To date, 19 different S100 proteins have been assigned to this protein family, and they show different degrees of similarity, ranging from 25 to 56% identity at the amino acid level. With S100B, S100P, and S100Z being the exceptions, the majority of the S100 genes are clustered on human chromosome 1q21 (33). Most S100 proteins serve as calcium sensor proteins that, upon activation, regulate the function and/or subcellular distribution of specific target proteins (13, 33, 47), and they are characterized by common structural motifs, including two low-affinity (K_D [equilibrium dissociation constant] of ∼10 μM to 100 μM) helix-loop-helix calcium binding domains (EF hands) that are separated by a hinge region and flanked by amino- and carboxy-terminal domains. The carboxy-terminal domain is variable among S100 proteins, and it typically is the site that is responsible for the selective interaction of each individual S100 protein with specific target proteins (30). S100 proteins are often upregulated in cancers, in inflammation, and in response to cellular stress (14, 16), suggesting that they function in cell responses to stress situations. Consistent with this hypothesis, stress situations were necessary to reveal phenotypes associated with the S100 knockout in mice (11, 14, 33, 56). Moreover, recent observations revealed a new function for the S100 protein family that included their ability to assist and regulate multichaperone complex-ligand interactions (41, 50, 51).One member of the S100 protein family, S100B, has attracted much interest in the past few years because, like other proteins implicated in neurodegeneration (e.g., amyloid, superoxide dismutase, and dual-specificity tyrosine phosphorylation-regulated kinase 1A), its gene is located within a segment of chromosome 21, which is trisomic in Down''s syndrome (DS). Its expression in the brain of mammals coincides with defined periods of central nervous system (CNS) maturation and cell differentiation (43). In oligodendrocyte progenitor cells (OPC), S100B expression is associated with differentiation, and S100B contributes to OPC differentiation in response to demyelinating insult (11). To understand the contribution of S100B to OPC differentiation, we searched for high-affinity S100B target proteins in this cell type by using far-Western analysis. A major and highly specific S100B target protein was identified, the mitochondrial ATAD3A protein.ATAD3A belongs to a new family of eukaryote-specific mitochondrial AAA⁺ ATPase proteins (17). In the human genome, two genes, Atad3A and Atad3B, are located in tandem on chromosome 1p36.33. The Atad3A gene is ubiquitous among multicellular organisms but absent in yeast. The Atad3B gene is specific to the human genome (27). ATAD3A is a mitochondrial protein anchored into the mitochondrial inner membrane (IM) at contact sites with the outer membrane (OM). Thanks to its simultaneous interaction with the two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the inner and outer membranes and controls diverse cell responses ranging from mitochondrial metabolism, cell growth, and mitochondrial fission 20a, 25). The ATAD3A protein has also been identified as a mitochondrial DNA binding protein (23) and as a cell surface antigen in some human tumors (20, 21). The plasma membrane localization of ATAD3A in tumor cells is suggestive that ATAD3A mitochondrial routing can be compromised in pathological situations such as cancer. To understand the functional response resulting from the interaction between S100B and ATAD3A, we first characterized the minimal interaction domain on ATAD3A for S100B binding using thermodynamic studies of wild-type and ATAD3A variants as well as via nuclear magnetic resonance (NMR) spectroscopy techniques. These studies allowed us to further refine the consensus S100B binding motif, which is conserved in several other S100B target proteins, including the p53 protein and several newly discovered target proteins associated with the cell translational machinery. We next analyzed the cellular interaction of S100B with truncated ATAD3A mutants that harbor the S100B binding domain but that are deficient for mitochondrial import. These studies revealed that S100B could assist ATAD3A mutant proteins during cytoplasmic processing by preventing dysfunctional aggregation events. Our results are discussed in light of the possible function of S100B in assisting the cytoplasmic processing of proteins for proper folding and subcellular localization.     

Global conservation priorities for marine turtles

Where conservation resources are limited and conservation targets are diverse, robust yet flexible priority-setting frameworks are vital. Priority-setting is especially important for geographically widespread species with distinct populations subject to multiple threats that operate on different spatial and temporal scales. Marine turtles are widely distributed and exhibit intra-specific variations in population sizes and trends, as well as reproduction and morphology. However, current global extinction risk assessment frameworks do not assess conservation status of spatially and biologically distinct marine turtle Regional Management Units (RMUs), and thus do not capture variations in population trends, impacts of threats, or necessary conservation actions across individual populations. To address this issue, we developed a new assessment framework that allowed us to evaluate, compare and organize marine turtle RMUs according to status and threats criteria. Because conservation priorities can vary widely (i.e. from avoiding imminent extinction to maintaining long-term monitoring efforts) we developed a “conservation priorities portfolio” system using categories of paired risk and threats scores for all RMUs (n = 58). We performed these assessments and rankings globally, by species, by ocean basin, and by recognized geopolitical bodies to identify patterns in risk, threats, and data gaps at different scales. This process resulted in characterization of risk and threats to all marine turtle RMUs, including identification of the world''s 11 most endangered marine turtle RMUs based on highest risk and threats scores. This system also highlighted important gaps in available information that is crucial for accurate conservation assessments. Overall, this priority-setting framework can provide guidance for research and conservation priorities at multiple relevant scales, and should serve as a model for conservation status assessments and priority-setting for widespread, long-lived taxa.     

Structural basis for the interaction of protein S1 with the Escherichia coli ribosome

In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding centre. In sharp contrast to its functional importance, S1 is still lacking from the high-resolution structures available for Escherichia coli and Thermus thermophilus ribosomes and thus the molecular mechanism governing the S1–ribosome interaction has still remained elusive. Here, we present the structure of the N-terminal S1 domain D1 when bound to the ribosome at atomic resolution by using a combination of NMR, X-ray crystallography and cryo-electron microscopy. Together with biochemical assays, the structure reveals that S1 is anchored to the ribosome primarily via a stabilizing π-stacking interaction within the short but conserved N-terminal segment that is flexibly connected to domain D1. This interaction is further stabilized by salt bridges involving the zinc binding pocket of protein S2. Overall, this work provides one hitherto enigmatic piece in the ′ribosome puzzle′, namely the detailed molecular insight into the topology of the S1–ribosome interface. Moreover, our data suggest novel mechanisms that have the potential to modulate protein synthesis in response to environmental cues by changing the affinity of S1 for the ribosome.     

Calcineurin B-like domains in the large regulatory alpha/beta subunits of phosphorylase kinase

Phosphorylase kinase (PhK) is a large hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase (GP). It consists in four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha beta delta). Delta corresponds to endogenous calmodulin, whereas little is known on the molecular architecture of the large alpha and beta subunits, which probably arose from gene duplication. Here, using sensitive methods of sequence analysis, we show that the C-terminal domain (named domain D) of these alpha and beta subunits can be significantly related to calcineurin B-like (CBL) proteins. CBL are members of the EF-hand family that are involved in the regulation of plant-specific kinases of the CIPK/PKS family, and relieve autoinhibition of their target kinases by binding to their regulatory region. The relationship highlighted here suggests that PhK alpha and/or beta domain D may be involved in a similar regulation mechanism, a hypothesis which is supported by the experimental observation of a direct interaction between domain D of PhKalpha and the regulatory region of the Gamma subunit. This finding, together the identification of significant similarities of domain D with the preceding domain C, may help to understand the molecular mechanism by which PhK alpha and/or beta domain D might regulate PhK activity.     

Irreversible inhibition of aldolase by a phosphorylated alpha-dicarbonyl compound

The preparation of a phosphorylated alpha-dicarbonyl compound designed to specifically react with arginine residues of enzymes accepting phosphorylated compounds as effectors is reported, and shown to inhibit rabbit muscle aldolase in a time-dependent and irreversible manner. This irreversible inhibition occured in a buffer devoid of borate ions, suggesting that the presence of the phosphate moiety contributes in the stabilization of the adduct formed with arginine residues. Under the same conditions, the metalloenzyme iron superoxide dismutase, in which an arginine is known to be critical for the catalytic function, is not significantly inhibited.