S92 phosphorylation induces structural changes in the N-terminus domain of human mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) plays essential roles in mitochondrial calcium homeostasis and regulates cellular functions, such as energy synthesis, cell growth, and development. Thus, MCU activity is tightly controlled by its regulators as well as post-translational modification, including phosphorylation by protein kinases such as proline-rich tyrosine kinase 2 (Pyk2) and AMP-activated protein kinase (AMPK). In our in vitro kinase assay, the MCU N-terminal domain (NTD) was phosphorylated by protein kinase C isoforms (PKCßII, PKCδ, and PKCε) localized in the mitochondrial matrix. In addition, we found the conserved S92 was phosphorylated by the PKC isoforms. To reveal the structural effect of MCU S92 phosphorylation (S92p), we determined crystal structures of the MCU NTD of S92E and D119A mutants and analysed the molecular dynamics simulation of WT and S92p. We observed conformational changes of the conserved loop2-loop4 (L2-L4 loops) in MCU NTDS92E, NTDD119A, and NTDS92p due to the breakage of the S92-D119 hydrogen bond. The results suggest that the phosphorylation of S92 induces conformational changes as well as enhancements of the negative charges at the L2-L4 loops, which may affect the dimerization of two MCU-EMRE tetramers.

Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

miR-185 plays an anti-hypertrophic role in the heart via multiple targets in the calcium-signaling pathways.

MicroRNA (miRNA) is an endogenous non-coding RNA species that either inhibits RNA translation or promotes degradation of target mRNAs. miRNAs often regulate cellular signaling by targeting multiple genes within the pathways. In the present study, using Gene Set Analysis, a useful bioinformatics tool to identify miRNAs with multiple target genes in the same pathways, we identified miR-185 as a key candidate regulator of cardiac hypertrophy. Using a mouse model, we found that miR-185 was significantly down-regulated in myocardial cells during cardiac hypertrophy induced by transverse aortic constriction. To confirm that miR-185 is an anti-hypertrophic miRNA, genetic manipulation studies such as overexpression and knock-down of miR-185 in neonatal rat ventricular myocytes were conducted. The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart. Our study further identified Camk2d, Ncx1, and Nfatc3 as direct targets of miR-185. The activity of Nuclear Factor of Activated T-cell (NFAT) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) was negatively regulated by miR-185 as assessed by NFAT-luciferase activity and western blotting. The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185. In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.

Coupling of ryanodine receptor 2 and voltage-dependent anion channel 2 is essential for Ca²+ transfer from the sarcoplasmic reticulum to the mitochondria in the heart.

The structural proximity and functional coupling between the SR (sarcoplasmic reticulum) and mitochondria have been suggested to occur in the heart. However, the molecular architecture involved in the SR-mitochondrial coupling remains unclear. In the present study, we performed various genetic and Ca2+-probing studies to resolve the proteins involved in the coupling process. By using the bacterial 2-hybrid, glutathione transferase pull-down, co-immunoprecipitation and immunocytochemistry assays, we found that RyR2 (ryanodine receptor type 2), which is physically associated with VDAC2 (voltage-dependent anion channel 2), was co-localized in SR-mitochondrial junctions. Furthermore, a fractionation study revealed that VDAC2 was co-localized with RyR2 only in the subsarcolemmal region. VDAC2 knockdown by targeted short hairpin RNA led to an increased diastolic [Ca2+] (calcium concentration) and abolishment of mitochondrial Ca2+ uptake. Collectively, the present study suggests that the coupling of VDAC2 with RyR2 is essential for Ca2+ transfer from the SR to mitochondria in the heart.

Increased store-operated Ca2+ entry in skeletal muscle with reduced calsequestrin-1 expression.

Store-operated Ca(2+) entry (SOCE) contributes to Ca(2+) handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn(2+) fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca(2+) store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39 degrees C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca(2+) permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca(2+) as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca(2+) entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca(2+) signaling in MH.