Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle.

Tropomodulin (E-Tmod) is an actin filament pointed end capping protein that maintains the length of the sarcomeric actin filaments in striated muscle. Here, we describe the identification and characterization of a novel tropomodulin isoform, skeletal tropomodulin (Sk-Tmod) from chickens. Sk-Tmod is 62% identical in amino acid sequence to the previously described chicken E-Tmod and is the product of a different gene. Sk-Tmod isoform sequences are highly conserved across vertebrates and constitute an independent group in the tropomodulin family. In vitro, chicken Sk-Tmod caps actin and tropomyosin-actin filament pointed ends to the same extent as does chicken E-Tmod. However, E- and Sk-Tmods differ in their tissue distribution; Sk-Tmod predominates in fast skeletal muscle fibers, lens, and erythrocytes, while E-Tmod is found in heart and slow skeletal muscle fibers. Additionally, their expression is developmentally regulated during chicken breast muscle differentiation with Sk-Tmod replacing E-Tmod after hatching. Finally, in skeletal muscle fibers that coexpress both Sk- and E-Tmod, they are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively. All together, these observations support the hypothesis that vertebrates have acquired different tropomodulin isoforms that play distinct roles in vivo.     

Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins

The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton. In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals. Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind alpha-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulin-dependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification. Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers. Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins alpha-actinin, gamma-filamin, and telethonin. In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc. Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.     

MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC

Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.     

Dysbindin, a novel coiled-coil-containing protein that interacts with the dystrobrevins in muscle and brain

The dystrophin-associated protein complex (DPC) is required for the maintenance of muscle integrity during the mechanical stresses of contraction and relaxation. In addition to providing a membrane scaffold, members of the DPC such as the alpha-dystrobrevin protein family are thought to play an important role in intracellular signal transduction. To gain additional insights into the function of the DPC, we performed a yeast two-hybrid screen for dystrobrevin-interacting proteins. Here we describe the identification of a dysbindin, a novel dystrobrevin-binding protein. Dysbindin is an evolutionary conserved 40-kDa coiled-coil-containing protein that binds to alpha- and beta-dystrobrevin in muscle and brain. Dystrophin and alpha-dystrobrevin are co-immunoprecipitated with dysbindin, indicating that dysbindin is DPC-associated in muscle. Dysbindin co-localizes with alpha-dystrobrevin at the sarcolemma and is up-regulated in dystrophin-deficient muscle. In the brain, dysbindin is found primarily in axon bundles and especially in certain axon terminals, notably mossy fiber synaptic terminals in the cerebellum and hippocampus. These findings have implications for the molecular pathology of Duchenne muscular dystrophy and may provide an alternative route for anchoring dystrobrevin and the DPC to the muscle membrane.     

The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.     

p38 MAPK interacts with actin and modulates filament assembly during skeletal muscle differentiation

Skeletal muscle differentiation is marked by enhanced myotube formation and increased cytoskeletal rearrangement. Actin, a cytoskeletal protein is involved in various cellular functions such as glucose transport, intracellular trafficking, cell shape, and coordinated cell movement in response to various extracellular signals. The present study reveals an association between actin and p38 MAPK only in differentiated myotubes, not in proliferating myoblasts. Actin filament disassembly caused by cytochalasinD can be reversed using the potent activator of p38 MAPK, anisomycin. Pretreatment of myotubes with anisomycin partially resisted the effect of cytochalasinD. However, inhibition of p38 MAPK completely abolished the anisomycin-mediated actin remodeling. Data suggests that p38 MAPK interacts with actin and modulates actin filament rearrangement in differentiated L6E9 skeletal muscle cells.     

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.     

MAGE-F1, a novel ubiquitously expressed member of the MAGE superfamily

Most known members of the MAGE superfamily are expressed in tumors, testis and fetal tissues, which has been described as a cancer/testis or "CT" expression pattern. We have identified a novel member of this superfamily, MAGE-F1, which is expressed in all adult and fetal tissues tested. In addition to normal tissues, MAGE-F1 is expressed in many tumor types including ovarian, breast, cervical, melanoma and leukemia. MAGE-F1 is encoded on chromosome 3, identifying a sixth chromosomal location for a MAGE superfamily gene. The coding region of MAGE-F1 is contained within a single exon and includes a microsatellite repeat. Sequence analysis and expression profiles define a new class of ubiquitously expressed MAGE superfamily genes that includes MAGE-F1, MAGE-D1, MAGE-D2/JCL-1 and NDN. The finding that several MAGE genes are ubiquitously expressed suggests a role for MAGE encoded proteins in normal cell physiology. Furthermore, potential cross-reactivity to these ubiquitously expressed MAGE gene products should be considered in the design of MAGE-targeted immunotherapies for cancer.     

BH-protocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain

Using a yeast two-hybrid system, we isolated eight cDNA clones which interacted with BH-protocadherin-c (BH-Pcdh-c) from the human brain cDNA library. One clone encoded protein phosphatase type I isoform alpha (PP1alpha) and another two PP1alpha2. PP1alpha was co-immunoprecipitated from the extract of a gastric adenocarcinoma cell line MKN-28 with anti-BH-Pcdh-c antibody. PP1alpha activity towards glycogen phosphorylase was inhibited by the intracellular domain of BH-Pcdh-c. Inhibition of the phosphatase required more than the minimal domain of BH-Pcdh-c which could associate with PP1alpha. In situ hybridization revealed that BH-Pcdh-c mRNA was predominantly expressed in cerebral cortex neurons in the adult mouse brain.     

Differential expression and developmental regulation of a novel alpha-dystrobrevin isoform in muscle

Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.     

Crystal structure of brain pyridoxal kinase,a novel member of the ribokinase superfamily

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 A, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B(6) and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp(118) and Tyr(127) in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.     

Cloning and biological comparison of Restin, a novel member of Mage superfamily

In the present study, a new member of melanoma associated antigens (Mage), named Restin (219 amino acids), was identified from HL-60 cell induced by all-trans-retinoic acid (ATRA) by PCR-based subtractive hybridization. Bioinformatics analysis found this novel gene shares high homolog with Necdin (a neuronal growth suppressor, 49%). Both of them are basic proteins. Moreover, the Restin, Necdin and Mages are in one protein superfamily. This fact indicates that the Restin and Mages are mutually related but functionally different. Further analysis found that they can be divided into two subgroups, the acid and the basic. Restin, Necdin and Mage-D1 have an alkaline conserve region (PI is from 8.6 to 10.1), which are not or less expressed in tumor tissues but mostly in normal tissues. It has been reported that Necdin can arrest the cell proliferation by interaction with p53 and E2F1. Therefore, all of them are probably related to arrest the cell cycle. However, the Mage A and C are primarily acid proteins (PI is from 4.2 to 4.9), not expressed in normal tissues but in tumors. It is quite probable that these proteins are involved in the cell proliferation. We therefore suggest that these two protein families might be a pair of control elements of cell cycle——“in cycle or out of cycle”.     

Cloning and biological comparison of Restin, a novel member of Mage superfamily

In the present study, a new member of melanoma associated antigens (Mage), named Restin (219 amino acids), was identified from HL-60 cell induced by all-trans-retinoic acid (ATRA) by PCR-based subtractive hybridization. Bioinformatics analysis found this novel gene shares high homolog with Necdin (a neuronal growth suppressor, 49%). Both of them are basic proteins. Moreover, the Restin, Necdin and Mages are in one protein superfamily. This fact indicates that the Restin and Mages are mutually related but functionally different. Further analysis found that they can be divided into two subgroups, the acid and the basic. Restin, Necdin and Mage-D1 have an alkaline conserve region (PI is from 8.6 to 10.1), which are not or less expressed in tumor tissues but mostly in normal tissues. It has been reported that Necdin can arrest the cell proliferation by interaction with p53 and E2F1. Therefore, all of them are probably related to arrest the cell cycle. However, the Mage A and C are primarily acid proteins (PI is from 4.2 to 4.9), not expressed in normal tissues but in tumors. It is quite probable that these proteins are involved in the cell proliferation. We therefore suggest that these two protein families might be a pair of control elements of cell cycle--"in cycle or out of cycle".     

HuBMSC-MCP, a novel member of mitochondrial carrier superfamily, enhances dendritic cell endocytosis

A novel member of mitochondrial carrier superfamily has been identified from human bone marrow stromal cells (BMSC) and designated as human BMSC-derived mitochondrial carrier protein (HuBMSC-MCP). It encodes a 321 amino-acid protein with three tandem related domains of about 100 amino acids. Each domain contains two hydrophobic stretches, which are thought to span the membrane as alpha-helices. Distant relationship analysis indicates that the protein is highly conserved between species from Caenorhabditis elegans to human. HuBMSC-MCP gene is mapped to chromosome 11p11. HuBMSC-MCP mRNA expression is detectable in various human tissues and cell lines. By confocal imaging, HuBMSC-MCP is localized to mitochondria and also detected in the pseudopodial protrusion of human breast adenocarcinoma MCF-7 cells. When transfected into dendritic cells (DC), HuBMSC-MCP could enhance DCs endocytotic capacity. Thus, HuBMSC-MCP is a phylogenetically conserved and widely expressed mitochondrial carrier protein which perhaps associates with mitochondrial oxidative phosphorylation.     

Cloning and biological comparison of Restin, a novel member of Mage superfamily

In the present study, a new member of melanoma associated antigens (Mage), named Restin (219 amino acids), was identified from HL-60 cell induced by all-frans-retinoic acid (ATRA) by PCR-based subtractive hybridization. Bioinformatics analysis found this novel gene shares high homolog with Necdin (a neuronal growth suppressor, 49%). Both of them are basic proteins. Moreover, the Restin, Necdin and Mages are in one protein superfamily. This fact indicates that the Restin and Mages are mutually related but functionally different. Further analysis found that they can be divided into two subgroups, the acid and the basic. Restin, Necdin and Mage-D1 have an alkaline conserve region (PI is from 8.6 to 10.1), which are not or less expressed in tumor tissues but mostly in normal tissues. It has been reported that Necdin can arrest the cell proliferation by interaction with p53 and E2F1. Therefore, all of them are probably related to arrest the cell cycle. However, the Mage A and C are primarily acid proteins (PI