Validation and classification of RNA binding proteins identified by mRNA interactome capture.

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

TRIM24 protein promotes and TRIM32 protein inhibits cardiomyocyte hypertrophy via regulation of dysbindin protein levels.

We have previously shown that dysbindin is a potent inducer of cardiomyocyte hypertrophy via activation of Rho-dependent serum-response factor (SRF) signaling. We have now performed a yeast two-hybrid screen using dysbindin as bait against a cardiac cDNA library to identify the cardiac dysbindin interactome. Among several putative binding proteins, we identified tripartite motif-containing protein 24 (TRIM24) and confirmed this interaction by co-immunoprecipitation and co-immunostaining. Another tripartite motif (TRIM) family protein, TRIM32, has been reported earlier as an E3 ubiquitin ligase for dysbindin in skeletal muscle. Consistently, we found that TRIM32 also degraded dysbindin in neonatal rat ventricular cardiomyocytes as well. Surprisingly, however, TRIM24 did not promote dysbindin decay but rather protected dysbindin against degradation by TRIM32. Correspondingly, TRIM32 attenuated the activation of SRF signaling and hypertrophy due to dysbindin, whereas TRIM24 promoted these effects in neonatal rat ventricular cardiomyocytes. This study also implies that TRIM32 is a key regulator of cell viability and apoptosis in cardiomyocytes via simultaneous activation of p53 and caspase-3/-7 and inhibition of X-linked inhibitor of apoptosis. In conclusion, we provide here a novel mechanism of post-translational regulation of dysbindin and hypertrophy via TRIM24 and TRIM32 and show the importance of TRIM32 in cardiomyocyte apoptosis in vitro.

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

Structural Basis of Neuronal Nitric-oxide Synthase Interaction with Dystrophin Repeats 16 and 17.

Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16-17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16-17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16-17 and molecular docking methods, we generated atomic models of the dystrophin R16-17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A' and a small fraction of helix B' and a large part of the helix C' of repeat R17. The interaction surface of nNOS-PDZ involves its main ß-sheet and its specific C-terminal ß-finger.

Disrupted-in-schizophrenia 1 (DISC1) regulates dysbindin function by enhancing its stability.

Dysbindin and DISC1 are schizophrenia susceptibility factors playing roles in neuronal development. Here we show that the physical interaction between dysbindin and DISC1 is critical for the stability of dysbindin and for the process of neurite outgrowth. We found that DISC1 forms a complex with dysbindin and increases its stability in association with a reduction in ubiquitylation. Furthermore, knockdown of DISC1 or expression of a deletion mutant, DISC1 lacking amino acid residues 403-504 of DISC1 (DISC1(Δ403-504)), effectively decreased levels of endogenous dysbindin. Finally, the neurite outgrowth defect induced by knockdown of DISC1 was partially reversed by coexpression of dysbindin. Taken together, these results indicate that dysbindin and DISC1 form a physiologically functional complex that is essential for normal neurite outgrowth.

Phosphorylation within the cysteine-rich region of dystrophin enhances its association with ß-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin.

Costamere proteins and their involvement in myopathic processes.

Muscle fibres are very specialised cells with a complex structure that requires a high level of organisation of the constituent proteins. For muscle contraction to function properly, there is a need for not only sarcomeres, the contractile structures of the muscle fibre, but also costameres. These are supramolecular structures associated with the sarcolemma that allow muscle adhesion to the extracellular matrix. They are composed of protein complexes that interact and whose functions include maintaining cell structure and signal transduction mediated by their constituent proteins. It is important to improve our understanding of these structures, as mutations in various genes that code for costamere proteins cause many types of muscular dystrophy. In this review, we provide a description of costameres detailing each of their constituent proteins, such as dystrophin, dystrobrevin, syntrophin, sarcoglycans, dystroglycans, vinculin, talin, integrins, desmin, plectin, etc. We describe as well the diseases associated with deficiency thereof, providing a general overview of their importance.

Syntrophin proteins as Santa Claus: role(s) in cell signal transduction.

Syntrophins are a family of cytoplasmic membrane-associated adaptor proteins, characterized by the presence of a unique domain organization comprised of a C-terminal syntrophin unique (SU) domain and an N-terminal pleckstrin homology (PH) domain that is split by insertion of a PDZ domain. Syntrophins have been recognized as an important component of many signaling events, and they seem to function more like the cell's own personal 'Santa Claus' that serves to 'gift' various signaling complexes with precise proteins that they 'wish for', and at the same time care enough for the spatial, temporal control of these signaling events, maintaining overall smooth functioning and general happiness of the cell. Syntrophins not only associate various ion channels and signaling proteins to the dystrophin-associated protein complex (DAPC), via a direct interaction with dystrophin protein but also serve as a link between the extracellular matrix and the intracellular downstream targets and cell cytoskeleton by interacting with F-actin. They play an important role in regulating the postsynaptic signal transduction, sarcolemmal localization of nNOS, EphA4 signaling at the neuromuscular junction, and G-protein mediated signaling. In our previous work, we reported a differential expression pattern of alpha-1-syntrophin (SNTA1) protein in esophageal and breast carcinomas. Implicated in several other pathologies, like cardiac dys-functioning, muscular dystrophies, diabetes, etc., these proteins provide a lot of scope for further studies. The present review focuses on the role of syntrophins in membrane targeting and regulation of cellular proteins, while highlighting their relevance in possible development and/or progression of pathologies including cancer which we have recently demonstrated.

Adiponectin receptor 1 C-terminus interacts with PDZ-domain proteins such as syntrophins.

Adiponectin receptor 1 (AdipoR1) is one of the two signaling receptors of adiponectin with multiple beneficial effects in metabolic diseases. AdipoR1 C-terminal peptide is concordant with the consensus sequence of class I PSD-95, disc large, ZO-1 (PDZ) proteins, and screening of a liver yeast two hybrid library identified binding to ß2-syntrophin (SNTB2). Hybridization of a PDZ-domain array with AdipoR1 C-terminal peptide shows association with PDZ-domains of further proteins including ß1- and α-syntrophin (SNTA). Interaction of PDZ proteins and C-terminal peptides requires a free carboxy terminus next to the PDZ-binding region and is blocked by carboxy terminal added tags. N-terminal tagged AdipoR1 is more highly expressed than C-terminal tagged receptor suggesting that the free carboxy terminus may form a complex with PDZ proteins to regulate cellular AdipoR1 levels. The C- and N-terminal tagged AdipoR1 proteins are mainly localized in the cytoplasma. N-terminal but not C-terminal tagged AdipoR1 colocalizes with syntrophins in adiponectin incubated Huh7 cells. Adiponectin induced hepatic phosphorylation of AMPK and p38 MAPK which are targets of AdipoR1 is, however, not blocked in SNTA and SNTB2 deficient mice. Further, AdipoR1 protein is similarly abundant in the liver of knock-out and wild type mice when kept on a standard chow or a high fat diet. In summary these data suggest that AdipoR1 protein levels are regulated by so far uncharacterized class I PDZ proteins which are distinct from SNTA and SNTB2.

Equilibrium unfolding of the PDZ domain of ß2-syntrophin.

ß2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic ß-cells. It contains a PDZ domain (ß2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of ß2-syntrophin allosterically regulates the affinity of ß2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of ß2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, ß2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of ß2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.

Design, synthesis, structure and binding properties of PDZ binding, cyclic beta-finger peptides.

Protein interaction domains (PIDs) play a critical role in signal transduction. One PID of great interest is the PDZ domain, a 100 amino-acid-residue domain. Most PDZ domains recognize short, C-terminal peptide motives. In the heterodimer of the nNOS-PDZ domain and the alpha-syntrophin-PDZ domain, however, one PDZ domain forms a beta-finger that binds to the other PDZ domain. We show here that cyclic peptides derived from the beta-finger of the nNOS-PDZ domain can bind the syntrophin-PDZ domain in the same manner as the whole domain. The structure of three "finger-peptides" of different size has been determined and the binding investigated using calorimetry and NMR-titration experiments.

Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat.

BACKGROUND: The dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain) is its distant relative, alpha-dystrobrevin. The alpha-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands) in the dystrophin glycoprotein complex. RESULTS: We show here that, contrary to the literature, most alpha-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse alpha-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms alpha-dystrobrevin-4 and -5) are present in most mammals but specifically ablated in mouse and rat. CONCLUSION: Lineage-specific mutations in the murids mean that the mouse brain has fewer than half of the alpha-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the alpha-dystrobrevins (and therefore the dystrophin glycoprotein complexes) of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins.

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.

Synaptic alpha-dystrobrevin: localization of a short alpha-dystrobrevin isoform in melanin-concentrating hormone neurons of the hypothalamus.

The expression of the two members of the dystrobrevin (DB) family in the adult brain was thought to be highly specific for the two main cell types: alpha-dystrobrevin (alpha-DB) and beta-dystrobrevin (beta-DB) has been identified as glial and neuronal proteins, respectively. In the present work we show that a subset of neurons in the hypothalamus contains alpha-DB. Comparative immunohistochemical studies with two alpha-DB antibodies of different specificity indicate that the neurons contain short alpha-DB isoform(s) alpha-DB-2 and/or alpha-DB-4. Immunoreactive multipolar or spindle-shaped neurons form clusters with bilateral symmetry, localized predominantly in the lateral hypothalamic area, with extensions into the zona incerta and the dorso-medial and ventro-medial hypothalamic region. alpha-DB immunoreactivity was localized in cell processes and at postsynaptic densities, furthermore in the endoplasmic reticulum within the perikarya. alpha-DB-positive neurons are beta-dystrobrevin immunoreactive, but alpha- and beta-DB do not co-localize with their usual molecular anchors like dystrophins or high molecular weight forms of utrophin. Colocalization with nNOS was also not observed. The pattern of alpha-DB immunoreactive neurons gave a perfect colocalization with melanin-concentrating hormone (MCH) neurons throughout the whole region studied. We propose that alpha-DB plays a role in a structure or regulation mechanism unique to MCH-expressing neurons.

Skeletal muscle syntrophin interactors revealed by yeast two-hybrid assay.

Syntrophins are the cytoplasmic peripheral proteins of dystrophin glycoprotein complex, of which five (alpha l, beta 1, beta 2, gamma 1 and gamma 2) isoforms have been identified so far. Respective syntrophin isoforms are encoded by different genes but have similar domain structures. At the sarcolemma of skeletal muscle, the most abundant alpha l-syntrophin was shown to interact at its PDZ domain with many membrane proteins. Among them, the AQP4 interaction with alpha 1-syntrophin PDZ domain was demonstrated by a Tg mouse study, prompting us to investigate the interaction between mouse alpha l-syntrophin (BC018546: nt.267-492, PDZ domain) pEXP-AD502 as prey vector and mouse AQP4 (NM009700: nt.805-969) pDBLeu as bait vector by the yeast two-hybrid assay, resulting in a negative study. We further studied the binding partner of another sarcolemma located beta 1-syntrophin, and performed a yeast two-hybrid experiment. With human beta 1-syntrophin as bait and human skeletal muscle cDNA library as prey, we obtained one positive clone which turned out to be alpha-dystrobrevin. Although the interaction of human beta 1-syntrophin with alpha-dystrobrevin has already been shown by immunoprecipitation assay, we have here confirmed this interaction by a yeast two-hybrid experiment.

Dystrophin R16/17-syntrophin PDZ fusion protein restores sarcolemmal nNOSµ.

BACKGROUND: Loss of sarcolemmal nNOSµ is a common manifestation in a wide variety of muscle diseases and contributes to the dysregulation of multiple muscle activities. Given the critical role sarcolemmal nNOSµ plays in muscle, restoration of sarcolemmal nNOSµ should be considered as an important therapeutic goal. METHODS: nNOSµ is anchored to the sarcolemma by dystrophin spectrin-like repeats 16 and 17 (R16/17) and the syntrophin PDZ domain (Syn PDZ). To develop a strategy that can independently restore sarcolemmal nNOSµ, we engineered an R16/17-Syn PDZ fusion construct and tested whether this construct alone is sufficient to anchor nNOSµ to the sarcolemma in three different mouse models of Duchenne muscular dystrophy (DMD). RESULTS: Membrane-associated nNOSµ is completely lost in DMD. Adeno-associated virus (AAV)-mediated delivery of the R16/17-Syn PDZ fusion construct successfully restored sarcolemmal nNOSµ in all three models. Further, nNOS restoration was independent of the dystrophin-associated protein complex. CONCLUSIONS: Our results suggest that the R16/17-Syn PDZ fusion construct is sufficient to restore sarcolemmal nNOSµ in the dystrophin-null muscle.

The dystrotelin, dystrophin and dystrobrevin superfamily: new paralogues and old isoforms.

BACKGROUND: Dystrophins and dystrobrevins are distantly related proteins with important but poorly understood roles in the function of metazoan muscular and neuronal tissues. Defects in them and their associated proteins cause a range of neuromuscular disorders. Members of this superfamily have been discovered in a relatively serendipitous way; we set out to compile a comprehensive description of dystrophin- and dystrobrevin-related sequences from available metazoan genome sequences, validated in representative organisms by RT-PCR, or acquired de novo from key species. RESULTS: Features of the superfamily revealed by our survey include: a) Dystrotelin, an entirely novel branch of the superfamily, present in most vertebrates examined. Dystrotelin is expressed in the central nervous system, and is a possible orthologue of Drosophila DAH. We describe the preliminary characterisation of its function, evolution and expression. b) A novel vertebrate member of the dystrobrevin family, gamma-dystrobrevin, an ancient branch now extant only in fish, but probably present in our own ancestors. Like dystrophin, zebrafish gamma-dystrobrevin mRNA is localised to myosepta. c) The extent of conservation of alternative splicing and alternative promoter use in the dystrophin and dystrobrevin genes; alternative splicing of dystrophin exons 73 and 78 and alpha-dystrobrevin exon 13 are conserved across vertebrates, as are the use of the Dp116, Dp71 and G-utrophin promoters; the Dp260 and Dp140 promoters are tetrapod innovations. d) The evolution of the unique N-terminus of DRP2 and its relationship to Dp116 and G-utrophin. e) A C-terminally truncated common ancestor of dystrophin and utrophin in cyclostomes. f) A severely restricted repertoire of dystrophin complex components in ascidians. CONCLUSION: We have refined our understanding of the evolutionary history and isoform diversity of the five previously reported vertebrate superfamily members and describe two novel members, dystrotelin and gamma-dystrobrevin. Dystrotelins, dystrophins and dystrobrevins are roughly equally related to each other. Vertebrates therefore have a repertoire of seven superfamily members (three dystrophins, three dystrobevins, and one dystrotelin), with one lost in tetrapods. Most invertebrates studied have one member from each branch. Although the basic shared function which is implied by the common architecture of these distantly related proteins remains unclear, it clearly permeates metazoan biology.

Dystrobrevins in muscle and non-muscle tissues.

The alpha- and beta-dystrobrevins belong to the family of dystrophin-related and dystrophin-associated proteins. As constituents of the dystrophin-associated protein complex, alpha-dystrobrevin was believed to have a role predominantly in muscles and beta-dystrobrevin in non-muscle tissues. Recent reports described novel localisations and molecular characteristics of alpha-dystrobrevin isoforms in non-muscle tissues (developing and adult). While single and double knockout studies have revealed distinct functions of dystrobrevin in some tissues, these also suggested a strong compensatory mechanism, where dystrobrevins displaying overlapping tissue expression pattern and structure/function similarity can substitute each other. No human disease has been unequivocally associated within mutations of dystrobrevin genes. However, some significant exceptions to these overlapping expression patterns, mainly in the brain, suggest that dystrobrevin mutations might underlie some specific motor, behavioural or cognitive defects. Dystrobrevin binding partner DTNBP1 (dysbindin) is a probable susceptibility gene for schizophrenia and bipolar affective disorder in some populations. As dysbindin abnormality is linked to Hermansky-Pudlak syndrome, dystrobrevins and/or their binding partners may also be required for proper function of other non-muscle tissues.

Molecular basis of dystrobrevin interaction with kinesin heavy chain: structural determinants of their binding.

Dystrobrevins are a family of widely expressed dystrophin-associated proteins that comprises alpha and beta isoforms and displays significant sequence homology with several protein-binding domains of the dystrophin C-terminal region. The complex distribution of the multiple dystrobrevin isoforms suggests that the variability of their composition may be important in mediating their function. We have recently identified kinesin as a novel dystrobrevin-interacting protein and localized the dystrobrevin-binding site on the cargo-binding domain of neuronal kinesin heavy chain (Kif5A). In the present study, we assessed the kinetics of the dystrobrevin-Kif5A interaction by quantitative pull-down assay and surface plasmon resonance (SPR) analysis and found that beta-dystrobrevin binds to kinesin with high affinity (K(D) approximately 40 nM). Comparison of the sensorgrams obtained with alpha and beta-dystrobrevin at the same concentration of analyte showed a lower affinity of alpha compared to that of beta-dystrobrevin, despite their functional domain homology and about 70% sequence identity. Analysis of the contribution of single dystrobrevin domains to the interaction revealed that the deletion of either the ZZ domain or the coiled-coil region decreased the kinetics of the interaction, suggesting that the tertiary structure of dystrobrevin may play a role in regulating the interaction of dystrobrevin with kinesin. In order to understand if structural changes induced by post-translational modifications could affect dystrobrevin affinity for kinesin, we phosphorylated beta-dystrobrevin in vitro and found that it showed reduced binding capacity towards kinesin. The interaction between the adaptor/scaffolding protein dystrobrevin and the motor protein kinesin may play a role in the transport and targeting of components of the dystrophin-associated protein complex to specific sites in the cell, with the differences in the binding properties of dystrobrevin isoforms reflecting their functional diversity within the same cell type. Phosphorylation events could have a regulatory role in this context.

The syntrophin-dystrobrevin subcomplex in human neuromuscular disorders.

The syntrophins and alpha-dystrobrevin form a subcomplex with dystrophin at the skeletal muscle membrane, and are also highly concentrated at the neuromuscular synapse. Here we demonstrate that the different syntrophins and alpha-dystrobrevin isoforms have distinct expression patterns during human skeletal muscle development, and are differentially affected by loss of dystrophin anchorage and denervation in human neuromuscular disease. During normal fetal development, and in Duchenne muscular dystrophy and denervation disorders, alpha1-syntrophin and alpha-dystrobrevin are absent or markedly reduced at the sarcolemmal membrane. beta1-Syntrophin is the predominant syntrophin isoform expressed at the muscle membrane during development, and it undergoes upregulation in response to loss of alpha1-syntrophin in Duchenne muscular dystrophy and in denervation. Upregulation of beta1-syntrophin in neuromuscular disorders is associated with re-expression of the fetal nicotinic acetylcholine receptor gamma-subunit, cardiac actin, and neonatal myosin, suggesting reversion of muscle fibers to an immature phenotype. We show that denervation specifically affects expression of the syntrophin-dystrobrevin subcomplex and does not affect levels or localization of other members of the dystrophin-associated protein complex. Our results confirm that dystrophin is required for anchorage of the syntrophin-dystrobrevin subcomplex and suggest that expression of the syntrophin-dystrobrevin complex may be independently regulated through neuromuscular transmission.