Mitsugumin 53 attenuates the activity of sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the roles of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca²⁺-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca²⁺-uptake through SERCA1a (more than 35%) at micromolar Ca²⁺ but not at nanomolar Ca²⁺, suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.     

Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse

Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6^clft1 allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6^clft1 allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family. genesis 48:303–308, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of the Gene Encoding Human Sarcolipin (SLN), a Proteolipid Associated with SERCA1: Absence of Structural Mutations in Five Patients with Brody Disease

Sarcolipin (SLN) is a low-molecular-weight protein that copurifies with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca²⁺ATPase (SERCA1). Genomic DNA and cDNA encoding human sarcolipin (SLN) were isolated and characterized and theSLNgene was mapped to chromosome 11q22–q23. Human, rabbit, and mouse cDNAs encode a protein of 31 amino acids. Homology of SLN with phospholamban (PLN) suggests that the first 7 hydrophilic amino acids are cytoplasmic, the next 19 hydrophobic amino acids form a single transmembrane helix, and the last 5 hydrophilic amino acids are lumenal. The cytoplasmic and transmembrane sequences are not well conserved among the three species, but the lumenal sequence is highly conserved. Like SERCA1, SLN is highly expressed in rabbit fast-twitch skeletal muscle, but it is expressed to a lower extent in slow-twitch muscle and to an even lower extent in cardiac muscle, where SERCA2a and PLN are highly expressed. It is expressed in only trace amounts in pancreas and prostate.SLNandPLNgenes resemble each other in having two small exons, with their entire coding sequences lying in exon 2 and a large intron separating the two segments. Brody disease is an inherited disorder of skeletal muscle function, characterized by exercise-induced impairment of muscle relaxation. Mutations in theATP2A1gene encoding SERCA1 have been associated with the autosomal recessive inheritance of Brody disease in three families, but not with autosomal dominant inheritance of the disease. A search for mutations in theSLNgene in five Brody families, four of which were not linked toATP2A1,did not reveal any alterations in coding, splice junction or promoter sequences. The homozygous deletion of C438 in the coding sequence ofATP2A1in Brody disease family 3, leading to a frameshift and truncation following Pro₁₄₇in SERCA1, is the fourthATP2A1mutation to be associated with autosomal recessive Brody disease.     

Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca²⁺-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca²⁺ homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca²⁺ concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.     

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump,SERCA

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn⁴-Ala and Thr⁵-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.     

accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1

When wild-type zebrafish embryos are touched at 24 hours post-fertilization (hpf), they typically perform two rapid alternating coils of the tail. By contrast, accordion (acc) mutants fail to coil their tails normally but contract the bilateral trunk muscles simultaneously to shorten the trunk, resulting in a pronounced dorsal bend. Electrophysiological recordings from muscles showed that the output from the central nervous system is normal in mutants, suggesting a defect in muscles is responsible. In fact, relaxation in acc muscle is significantly slower than normal. In vivo imaging of muscle Ca2+ transients revealed that cytosolic Ca2+ decay was significantly slower in acc muscle. Thus, it appears that the mutant behavior is caused by a muscle relaxation defect due to the impairment of Ca2+ re-uptake. Indeed, acc mutants carry a mutation in atp2a1 gene that encodes the sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump found in the muscle sarcoplasmic reticulum (SR) that is responsible for pumping Ca2+ from the cytosol back to the SR. As SERCA1 mutations in humans lead to Brody disease, an exercise-induced muscle relaxation disorder, zebrafish accordion mutants could be a useful animal model for this condition.     

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G > A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.     

NaV1.6a is required for normal activation of motor circuits normally excited by tactile stimulation

A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non‐active (nav^mi89 and nav^mi130). nav embryos displayed diminished spontaneous and touch‐evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding Na_V1.6a (scn8aa) as a potential candidate for nav. Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in Na_V1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild‐type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav. In‐vivo electrophysiological analysis of the touch‐evoked escape circuit indicated that voltage‐dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that Na_V1.6a was required for touch‐induced activation of the swim locomotor network. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:508–522, 2010     

Cellular compartmentation of aromatic amino acids in Neurospora crassa. I. Occupation of a protein synthesis pool by phenylalanine in Tyr-1 mutants

The p-fluorophenylalanine (FPA) resistance of acc ^phe, which has previously been shown (Brooks et al., 1972) to be a try-1 mutant, has been further investigated. When incubated in the absence of tyrosine, acc ^phe and also tyr-1 auxotrophs show a gradual increase in free phenylalanine in the cell but a sharp decrease in FPA incorporation into protein. The decrease in FPA incorporation is apparently due to the excess phenylalanine in the mutants, since the normal endogenous pool component in wild type and also in the mutants incubated on tyrosine does not appear to compete with FPA for incorporation. The rate of FPA incorporation into protein in acc ^phe remains at 10–15% of the wild-type rate even when the ratio of free FPA to excess phenylalanine in the cell is high as 8:1. If wild type is supplied with exogenous phenylalanine and FPA simultaneously, phenylalanine is preferentially incorporated into protein but, in contrast to the mutant, the rate of FPA incorporation increases as the ratio of free FPA to phenylalanine increases. On the basis of differences in competition with FPA and in susceptibilities to mild extraction procedures, it is proposed that phenylalanine can be located in at least three compartments in Neurospora: a small constant-size endogenous pool always seen in wild type; an expandable exogenous pool; and a protein synthesis pool which is preferentially populated by endogenous phenylalanine but can be entered by exogenous molecules when biosynthesis is regulated. In acc ^phe, where phenylalanine biosynthesis is not regulated, the excess phenylalanine is located primarily in the protein synthesis pool where it only has to compete with a small FPA component and is thereby preferentially incorporated into protein in this mutant.This work was supported, in part, by an Atomic Energy Commission grant to the Institute of Molecular Biophysics, The Florida State University, and by the Genetics Training Grant, funded by the National Institutes of Health. It contains, in part, data from the doctoral thesis of the senior author, who was supported by a Florida State University Nuclear Fellowship and by a Public Health Service Fellowship.     

Unique biochemical and behavioral alterations in drosophilashibirets1 mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

ABSTRACT Dynamin is a GTPase protein that is essential for clathrin‐mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi^ts1 changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton‐X extraction. Addition of Na⁺ or K⁺ can extract dynamin to the cytosolic (supernatant) fraction. The shi^ts1 mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature‐sensitive alleles or wild type. Sensitivity to salt extraction in shi^ts1 was enhanced by GTP and nonhydrolyzable GTP‐γS. The shi^ts1 mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature‐sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi^ts1 flies recovered much more slowly from heat‐induced paralysis than did other temperature‐sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd^msf15), which reduces the paralytic temperature threshold of other temperature‐sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi^ts1. Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 319–329, 2002     

Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca2 + -ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca^2?+? release, and the light fraction enriched in Ca^2?+?-ATPase (SERCA), an enzyme responsible for Ca^2?+? transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca^2?+?- ATPase activity and Ca^2?+?-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca^2?+?-uptake and Ca^2?+?-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.     

Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^? allele. The promoter of both alleles contains the effector binding element (EBE_PthA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.     

Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

 The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35° C, but not at 37° C. A revertant of srm1-1 ded1-21 that became able to grow at 37° C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2. Received: 20 March 1996/Accepted: 1 July 1996     

Atypical fast SERCA1a protein expression in slow myofibers and differential S-nitrosylation prevented by exercise during long term bed rest

We monitored changes in SERCA isoform specific expression and S-nitrosylation in myofibers of lower limb soleus (SOL) and vastus lateralis (VL) muscle biopsies before and after 60 days of voluntary long term bed rest (BR) without (BR-CTRL group, n = 8) and with exercise countermeasure (BR-EX group, n = 8). Before BR, a typical myofiber type-specific distribution of fast and slow SERCA1/2a isoforms was seen. After BR, a subpopulation (approx. 15%) of slow myofibers in BR-CTRL additionally expressed the fast SERCA1a isoform which was not seen in BR-EX. After BR, SERCA1a S-nitrosylation patterns analyzed by the biotin-switch assay decreased in disused SOL only but increased in both muscles following exercise. Differential SERCA1a S-nitrosylation and SERCA1a/2a co-expression in subsets of slow myofibers should be considered as signs of an altered cytosolic Ca²⁺ homeostasis following chronic muscle disuse. Exercise preserved myofiber type-specific SERCA1a expression and S-nitrosylation in VL and SOL in a different way, suggesting muscle-specific responses to the countermeasure protocol applied during bed rest.     

The Purkinje cell degeneration 5J mutation is a single amino acid insertion that destabilizes Nna1 protein

In the mouse, Purkinje cell degeneration (pcd) is a recessive mutation characterized by degeneration of cerebellar Purkinje cells, retinal photoreceptors, olfactory bulb mitral neurons, and certain thalamic neurons, and is accompanied by defective spermatogenesis. Previous studies of pcd have led to the identification of Nna1 as the causal gene; however, how loss of Nna1 function results in neurodegeneration remains unresolved. One useful approach for establishing which functional domains of a protein underlie a recessive phenotype has been to determine the genetic basis of the various alleles at the locus of interest. Because none of the pcd alleles analyzed at the time of the identification of Nna1 provided insight into the molecular basis of Nna1 loss-of-function, we obtained a recent pcd remutation—pcd^5J, and after determining that its phenotype is comparable to existing pcd severe alleles, we sought its genetic basis by sequencing Nna1. In this article we report that pcd^5J results from the insertion of a single GAC triplet encoding an aspartic acid residue at position 775 of Nna1. Although this insertion does not affect Nna1 expression at the RNA level, Nna1^pcd-5J protein expression is markedly decreased. Pulse-chase experiments reveal that the aspartic acid insertion dramatically destabilizes Nna1^pcd-5J protein, accounting for the observation that pcd^5J is a severe allele. The presence of a readily detectable genetic mutation in pcd^5J confirms that Nna1 loss-of-function alone underlies the broad pcd phenotype and will facilitate further studies of how Nna1 loss-of-function produces neurodegeneration and defective spermatogenesis in pcd mice.     

Deneddylase 1 regulates deneddylase activity of the Cop9 signalosome in Drosophila melanogaster

NEDD8 conjugation of Cullin has an important role in ubiquitin‐mediated protein degradation. The COP9 signalosome, of which CSN5 is the major catalytic subunit, is a major Cullin deneddylase. Another deneddylase, Deneddylase 1, has also been shown to process the Nedd8 precursor. In Drosophila, the DEN1 mutants do not have increased levels of Cullin neddylation, but instead show a significant decrease in neddylated Cullin. This characteristic decrease in neddylated Cullins in the DEN1^null background can be rescued by UAS‐dDEN1^WT overexpression but not by overexpression of mature NEDD8, indicating that this phenotype is distinct from the NEDD8‐processing function of DEN1. We examined the role of DEN1–CSN interaction in regulating Cullin neddylation. Overexpression of DEN1 in a CSN5^hypo background slightly reduced unneddylated Cullin levels. The CSN5, DEN1 double mutation partially rescues the premature lethality associated with the CSN5 single mutation. These results suggest that DEN1 regulates Cullin neddylation by suppressing CSN deneddylase activity.     

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle

Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ∼2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca²⁺ affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca²⁺ in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca²⁺ misregulation.