Overlapping ATP2C1 and ASTE1 genes in human genome: Implications for SPCA1 expression

Abstract: The ATP2C1 gene encodes for the secretory pathway calcium (Ca 2+)-ATPase pump (SPCA1), which localizes along the secretory pathway, mainly in the trans-Golgi. The loss of one ATP2C1 allele causes Hailey-Hailey disease in humans but not mice. Examining differences in genomic organization between mouse and human we speculate that the overlap between ATP2C1 and ASTE1 genes only in humans could explain this different response to ATP2C1 dysregulation. We propose that ASTE1, overlapping with ATP2C1 in humans, affects alternative splicing, and potentially protein expression of the latter. If dysregulated, the composition of the SPCA1 isoform pool could diverge from the physiological status, affecting cytosolic Ca 2+-signaling, and in turn perturbing cell division, leading to cell death or to neoplastic transformation

3D-reconstruction of the structural association between triads and mitochondria in skeletal fibers

Skeletal muscle can vary considerably its metabolism during activity. Since cell metabolism is controlled by Ca2þ signals, the precise positioning of mitochondria next to the Ca2þ release sites may be of great importance for the proper functioning of muscle fibers. In adult skeletal muscle, mitochondria are mostly located in proximity of triads, structures formed by the close apposition of SR and Ttubules, which mediate excitation-contraction coupling releasing Ca2þ during muscle activation (calcium release units, CRUs). Using electron microscopy, we have identified short strands (or tethers, ~10 nm long) connecting specifically the outer membrane of mitochondria to the SR, on the opposite side to the T-tubules (Boncompagni and Protasi, 2007; Biophys J. 92:A313). Tethers frequency increases during post-natal maturation, suggesting that these structures may have a crucial role in the progressive targeting of mitochondria next to triads. The SR-mitochondria association is sufficiently strong that treatment of FDB fibers with hypotonic solution results in stretching of the SR vesicle in correspondence of tethers. Using electron tomography, we have reconstructed the tri-dimensional architecture of triad/mitochondria interface providing additional information on the structural relationship between these two myoplasmic organelles. SR surrounds the mitochondria with lateral sacks/tubules and it is closely associated to it (tethered) also in mitochondrial regions closer to the Z line, on the side opposite to the triad. The molecular nature of the physical linkage is not yet identified and for now we can only speculate as to the possible structural importance that these small bridges may have in holding SR and mitochondria together

Characterization and temporal development of cores in a mouse model of malignant hyperthermia

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca2+ release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1Y522S/WT), the mutation causes SR Ca2+ leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1Y522S/WT knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1Y522S/WT mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2–4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1Y522S/WT mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca2+ sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements

Selenite biotransformation and detoxification by Stenotrophomonas maltophilia SeITE02: Novel clues on the route to bacterial biogenesis of selenium nanoparticles

A putative biosynthetic mechanism for selenium nanoparticles (SeNPs) and efficient reduction of selenite (SeO32-) in the bacterial strain Stenotrophomonas maltophilia SeITE02 are addressed here on the basis of information gained by a combined approach relying on a set of physiological, chemical/biochemical, microscopy, and proteomic analyses. S. maltophilia SeITE02 is demonstrated to efficiently transform selenite into elemental selenium (Se°) by reducing 100% of 0.5mM of this toxic oxyanion to Se° nanoparticles within 48h growth, in liquid medium. Since the selenite reducing activity was detected in the cytoplasmic protein fraction, while biogenic SeNPs showed mainly extracellular localization, a releasing mechanism of SeNPs from the intracellular environment is hypothesized. SeNPs appeared spherical in shape and with size ranging from 160nm to 250nm, depending on the age of the cultures. Proteomic analysis carried out on the cytoplasmic fraction identified an alcohol dehydrogenase homolog, conceivably correlated with the biogenesis of SeNPs. Finally, by Fourier Transformed Infrared Spectrometry, protein and lipid residues were detected on the surface of biogenic SeNPs. Eventually, this strain might be efficaciously exploited for the remediation of selenite-contaminated environmental matrices due to its high SeO32- reducing efficiency. Biogenic SeNPs may also be considered for technological applications in different fields

ARTD15 is a Kapß1 interactor.

<p>(<b>A</b>) Lysates (6 mg protein) from HeLa cells transfected with empty vector (control) or FLAG-ARTD15 were immunoprecipitated with a polyclonal anti-FLAG antibody. Proteins were separated by 10% long SDS-PAGE and proteins revealed by silver staining. Differential proteins were excised from the gel and identified by MALDI-ToF mass spectrometry (boxed bands). (<b>B</b>) Cell lysates from HeLa cells (10⁶ cells/assay) transfected with FLAG-ARTD15 were immunoprecipitated with an anti-Kapß1 antibody or with control IgG. The input is shown (1/20 of the total sample). (<b>C</b>) GST or GST-ARTD15 (0.1 µM) were incubated with increasing amounts of His-Kapß1. GST proteins were pulled down with gluthathione resin. Precipitated Kapß1 protein was probed with an anti-His antibody. (<b>D</b>) Immunofluorescence staining of endogenous ARTD15 (green) and endogenous Kapß1 (red) in HeLa cells. Bar, 20 µm.</p

ARTD15 is an ER resident protein.

<p>HeLa cells were transfected with FLAG-ARTD15. (<b>A</b>) Immunofluorescence staining of ARTD15 (green) in combination with the indicated markers (red) shows co-localization of ARTD15 protein with the ER. Bar, 20 µm. (<b>B</b>) Electron microscopy analysis of immuno-gold staining of ARTD15 (black dots). Arrows show ER tubular structures and nuclear envelope; cellular organelles are indicated by abbreviations (n: nucleus; pm: plasma membrane; m: mitochondria; ne: nuclear envelope). Bar, 500 nM. Data shown are representative of three independent experiments.</p

ARTD15 is expressed in different human cell lines.

<p>(<b>A</b>) Schematic diagram of ARTD15 domain structure. CD: catalytic domain; TD: transmembrane domain. (<b>B</b>) Levels of ARTD15 transcript determined by qRT-PCR, normalized to GAPDH RNA and then reported as arbitrary units relative to the ARTD15 transcript in HK2 cells (taken as 1). Data shown represent the mean (±SD) of two independent experiments performed in triplicate. (<b>C</b>) Expression of ARTD15. Western blotting (WB) showing endogenous ARTD15 protein and actin levels in 50 µg protein from total cell lysates. The expression levels of the ARTD15, normalized to actin, are shown in the histogram relative to those of HEK293 cells (taken as 100). Data shown represent the mean (±SD) of two independent experiments performed in triplicate. (<b>D</b>) Ponceau S staining and Western blotting (WB) of 100 µg protein from total HEK293 cell lysate are shown. (<b>E</b>) Immunofluorescence staining of endogenous ARTD15 (green) in combination with PDI (red) shows co-localization of ARTD15 protein with the ER. Bar, 20 µm.</p

ARTD15 is an ER tail-anchored protein.

<p>HeLa cells were transiently transfected with full-length (ARTD15) or deleted (ΔTM-ARTD15) FLAG-ARTD15 and analyzed either by (<b>A</b>) immuno-fluorescence microscopy with an anti-FLAG antibody; Bar, 20 µm or by (<b>B</b>) Western blotting of total lysates (60 µg), cytosol and total membrane (30 µg) proteins using anti-FLAG antibody to visualize ARTD15 and anti-GRK2 and anti-calnexin antibodies as a control of cell fractionation. (<b>C</b>) Protease protection assay performed with HeLa cells transfected with N-termini or C-termini GFP-ARTD15. Western blotting of ARTD15 revealed with an anti-GFP antibody, and of calnexin revealed with antibodies raised against the N-termini or C-termini of calnexin. (<b>D</b>) Schematic representation of ARTD15 (based on our results) and calnexin protein orientation. The N-termini (N) and C-termini (C) of the proteins with respect to the endoplasmic reticulum are indicated. The data shown in A, B, and C are representative of at least three independent experiments.</p