Maysoon Salih

The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy

The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

The muscle-specific calmodulin-dependent protein kinase assembles with the glycolytic enzyme complex at the sarcoplasmic reticulum and modulates the activity of glyceraldehyde-3-phosphate dehydrogenase in a Ca2+/calmodulin-dependent manner

The skeletal muscle specific Ca2+/calmodulin-dependent protein kinase (CaMKIIβM) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, αKAP, but its function remains to be defined. Protein interactions of CaMKIIβM indicated that it exists in complex with enzymes involved in glycolysis at the SR membrane. The kinase was found to complex with glycogen phosphorylase, glycogen debranching enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and creatine kinase in the SR membrane. CaMKIIβM was also found to assemble with aldolase A, GAPDH, enolase, lactate dehydrogenase, creatine kinase, pyruvate kinase, and phosphorylase b kinase from the cytosolic fraction. The interacting proteins were substrates of CaMKIIβM, and their phosphorylation was enhanced in a Ca2+- and calmodulin (CaM)-dependent manner. The CaMKIIβM could directly phosphorylate GAPDH and markedly increase (∼3.4-fold) its activity in a Ca2+/CaM-dependent manner. These data suggest that the muscle CaMKIIβM isoform may serve to assemble the glycogen-mobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKIIβM in response to calcium signaling would serve to modulate GAPDH and thereby ATP and NADH levels at the SR membrane, which in turn will regulate calcium transport processes.

Refined physical map of the spinal muscular atrophy gene (SMA) region at 5q13 based on YAC and cosmid contiguous arrays

The gene for the autosomal recessive neurodegenerative disorder spinal muscular atrophy has been mapped to a region of 5q13 flanked proximally by CMS-1 and distally by D5S557. We present a 2-Mb yeast artificial chromosome (YAC) contig constructed from three libraries encompassing the D5S435/D5S629/CMS-1-SMA-D5S557/D5S112 interval. The D5S629/CMS-1-SMA-D5S557 interval is unusual insofar as chromosome 5-specific repetitive sequences are present and many of the simple tandem repeats (STR) are located at multiple loci that are unstable in our YAC clones. A long-range restriction map that demonstrates the SMA-containing interval to be 550 kb is presented. Moreover, a 210-kb cosmid array from both a YAC-specific and a chromosome 5-specific cosmid library encompassing the multilocus STRs CATT-1, CMS-1, D5F149, D5F150, and D5F153 has been assembled. We have recently reported strong linkage disequilibrium with Type I SMA for two of these STRs, indicating that the gene is located in close proximity to or within our cosmid clone array.

A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre.

The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with γ-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.

MOLECULAR CLONING, EXPRESSION, AND CHROMOSOMAL ASSIGNMENT OF SARCOLEMMAL-ASSOCIATED PROTEINS : A FAMILY OF ACIDIC AMPHIPATHIC ALPHA -HELICAL PROTEINS ASSOCIATED WITH THE MEMBRANE

Two overlapping cDNAs encoding a novel sarcolemmal associated protein (SLAP) were isolated from a cardiac cDNA expression library by immunoscreening with anti-sarcolemmal antibodies. Further characterization of these clones showed that they belonged to a family of related cDNAs that potentially encode polypeptides of 37, 46, and 74 kDa designated SLAP1, SLAP2, and SLAP3, respectively. The SLAP3 transcript was ubiquitously expressed, whereas SLAP1 and SLAP2 transcripts were predominantly expressed in cardiac, soleus, and smooth muscle. SLAP was encoded by a single gene that mapped to chromosome 3p14.3–21.2, and the various transcripts are likely generated by alternative splicing. The primary structure of SLAP predicted that it would have large regions of coiled-coil structure including an 11-heptad acidic amphipathic α-helical segment. The carboxyl-terminal region of the SLAP proteins was predicted to have a transmembrane domain, although there was no discernible signal sequence. SLAPs could only be solubilized from cardiac membrane with detergents suggesting that they were integral membrane proteins. Subcellular distribution studies showed that MYC epitope-tagged SLAP localized to regions of juxtaposition between neighboring cell membranes although an intracellular pool of the protein was also present in cells undergoing apparent cleavage. Immunohistochemical localization of SLAP in cardiac muscle revealed that SLAP associated with the sarcolemma and also displayed a reticular pattern of staining that resembled the transverse tubules and the sarcoplasmic reticulum. The SLAPs define a new family of tail-anchored membrane proteins that exhibit tissue-specific expression and are uniquely situated to serve a variety of roles through their coiled-coil motifs.

Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

BackgroundTail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown.ResultsTA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1.ConclusionThus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

Regulated expression and temporal induction of the tail-anchored sarcolemmal-membrane-associated protein is critical for myoblast fusion

Sarcolemmal-membrane-associated proteins (SLMAPs) define a new class of coiled-coil tail-anchored membrane proteins generated by alternative splicing mechanisms. An in vivo expression analysis indicated that SLMAPs are present in somites (11 days post-coitum) as well as in fusing myotubes and reside at the level of the sarcoplasmic reticulum and transverse tubules in adult skeletal muscles. Skeletal-muscle myoblasts were found to express a single 5.9 kb transcript, which encodes the full-length approximately 91 kDa SLMAP3 isoform. Myoblast differentiation was accompanied by the stable expression of the approximately 91 kDa SLMAP protein as well as the appearance of an approximately 80 kDa isoform. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Membrane targeting of the de-regulated SLMAPs was not critical for the inhibition of myotube development. Protein-protein interaction assays indicated that SLMAPs are capable of self-assembling, and the de-regulated expression of mutants that were not capable of forming SLMAP homodimers also inhibited myotube formation. These results imply that regulated levels and the temporal induction of SLMAP isoforms are important for normal muscle development.

Tail-anchored membrane protein SLMAP is a novel regulator of cardiac function at the sarcoplasmic reticulum

Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dt(max)) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca(2+)-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function.

α-Kinase Anchoring Protein αKAP Interacts with SERCA2A to Spatially Position Ca2+/Calmodulin-dependent Protein Kinase II and Modulate Phospholamban Phosphorylation

The sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) is critical for sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR) and regulating cardiac muscle relaxation. Protein-protein interactions indicated that it exists in complex with Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its anchoring protein αKAP. Confocal imaging of isolated cardiomyocytes revealed the colocalization of CAMKII and αKAP with SERCA2a at the SR. Deletion analysis indicated that SERCA2a and CaMKII bind to different regions in the association domain of αKAP but not with each other. Although deletion of the putative N-terminal hydrophobic amino acid stretch in αKAP prevented its membrane targeting, it did not influence binding to SERCA2a or CaMKII. Both CaMKIIδC and the novel CaMKIIβ4 isoforms were found to exist in complex with αKAP and SERCA2a at the SR and were able to phosphorylate Thr-17 on phospholamban (PLN), an accessory subunit and known regulator of SERCA2a activity. Interestingly, the presence of αKAP was also found to significantly modulate the Ca2+/calmodulin-dependent phosphorylation of Thr-17 on PLN. These data demonstrate that αKAP exhibits a novel interaction with SERCA2a and may serve to spatially position CaMKII isoforms at the SR and to uniquely modulate the phosphorylation of PLN.

The E2F6 repressor activates gene expression in myocardium resulting in dilated cardiomyopathy

The E2F/Rb pathway regulates cardiac growth and development and holds great potential as a therapeutic target. The E2F6 repressor is a unique E2F member that acts independently of pocket proteins. Forced expression of E2F6 in mouse myocardium induced heart failure and mortality, with severity of symptoms correlating to E2F6 levels. Echocardiography demonstrated a 37% increase (P>0.05) in left ventricular end‐diastolic diameter and reduced ejection fraction (<40%, P>0.05) in young transgenic (Tg) mice. Microarray and qPCR analysis revealed a paradoxical increase in E2F‐responsive genes, which regulate the cell cycle, without changes in cardiomyocyte cell number or size in Tg mice. Young adult Tg mice displayed a 75% (P>0.01) decrease in gap junction protein connexin‐43, resulting in abnormal electrocardiogram including a 24% (P>0.05) increase in PR interval. Further, mir‐206, which targets connexin‐43, was up‐regulated 10‐fold (P>0.05) in Tg myocardium. The mitogen‐activated protein kinase pathway, which regulates the levels of miR‐206 and connexin‐43, was activated in Tg hearts. Thus, deregulated E2F6 levels evoked abnormal gene expression at transcriptional and post‐transcriptional levels, leading to cardiac remodeling and dilated cardiomyopathy. The data highlight an unprecedented role for the strict regulation of the E2F pathway in normal postnatal cardiac function.—Westendorp, B., Major, J. L., Nader, M., Salih, M., Leenen, F. H. H., Tuana, B. S. The E2F6 repressor activates gene expression in myocardium resulting in dilated cardiomyopathy. FASEB J. 26, 2569‐2579 (2012). www.fasebj.org