OBSCN restoration via OBSCN-AS1 long-noncoding RNA CRISPR-targeting suppresses metastasis in triple-negative breast cancer.

Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan-Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 (OBSCN-AS1), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN, and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids in vitro and metastasis in vivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.

Deletion of obscurin immunoglobulin domains Ig58/59 leads to age-dependent cardiac remodeling and arrhythmia.

Obscurin comprises a family of giant modular proteins that play key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin mediate binding to essential modulators of muscle structure and function, including canonical titin, a smaller splice variant of titin, termed novex-3, and phospholamban (PLN). Importantly, missense mutations localized within the obscurin-Ig58/59 region that affect binding to titins and/or PLN have been linked to the development of myopathy in humans. To elucidate the pathophysiological role of this region, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59, and determined the consequences of this manipulation on cardiac morphology and function under conditions of acute stress and through the physiological process of aging. Our studies show that young Obscn-ΔIg58/59 mice are susceptible to acute ß-adrenergic stress. Moreover, sedentary Obscn-ΔIg58/59 mice develop left ventricular hypertrophy that progresses to dilation, contractile impairment, atrial enlargement, and arrhythmia as a function of aging with males being more affected than females. Experiments in ventricular cardiomyocytes revealed altered Ca2+ cycling associated with changes in the expression and/or phosphorylation levels of major Ca2+ cycling proteins, including PLN, SERCA2, and RyR2. Taken together, our work demonstrates that obscurin-Ig58/59 is an essential regulatory module in the heart and its deletion leads to age- and sex-dependent cardiac remodeling, ventricular dilation, and arrhythmia due to deregulated Ca2+ cycling.

Novel mutations in MYBPC1 are associated with myogenic tremor and mild myopathy.

OBJECTIVE: To define a distinct, dominantly inherited, mild skeletal myopathy associated with prominent and consistent tremor in two unrelated, three-generation families. METHODS: Clinical evaluations as well as exome and panel sequencing analyses were performed in affected and nonaffected members of two families to identify genetic variants segregating with the phenotype. Histological assessment of a muscle biopsy specimen was performed in 1 patient, and quantitative tremor analysis was carried out in 2 patients. Molecular modeling studies and biochemical assays were performed for both mutations. RESULTS: Two novel missense mutations in MYBPC1 (p.E248K in family 1 and p.Y247H in family 2) were identified and shown to segregate perfectly with the myopathy/tremor phenotype in the respective families. MYBPC1 encodes slow myosin binding protein-C (sMyBP-C), a modular sarcomeric protein playing structural and regulatory roles through its dynamic interaction with actin and myosin filaments. The Y247H and E248K mutations are located in the NH2 -terminal M-motif of sMyBP-C. Both mutations result in markedly increased binding of the NH2 terminus to myosin, possibly interfering with normal cross-bridge cycling as the first muscle-based step in tremor genesis. The clinical tremor features observed in all mutation carriers, together with the tremor physiology studies performed in family 2, suggest amplification by an additional central loop modulating the clinical tremor phenomenology. INTERPRETATION: Here, we link two novel missense mutations in MYBPC1 with a dominant, mild skeletal myopathy invariably associated with a distinctive tremor. The molecular, genetic, and clinical studies are consistent with a unique sarcomeric origin of the tremor, which we classify as "myogenic tremor." ANN NEUROL 2019.

Sarcomeric myopathies associated with tremor: new insights and perspectives.

Myopathies are a large and heterogeneous group of disorders associated with mutations in structural and regulatory genes responsible for proper muscle assembly, organization and function. Despite the molecular diversity of inherited myopathies, they have historically been classified by the phenotypic traits observed in affected patients. It is therefore common for myopathies originating from mutations in different genes to be grouped together due to similar physical manifestations, and conversely myopathies resulting from mutations in the same gene to be considered separately due to disparate symptoms. Herein, we focus on an early onset myopathy linked to inherited or de novo mutations in sarcomeric genes that is characterized by muscle weakness, hypotonia and tremor, and further highlight that it may constitute a new form of myopathy, with tremor as its defining feature. Based on recent reports, we also discuss the possible myogenic origin of the tremor that may start at the level of the sarcomere due to structural and/or contractile alterations occurring as a result of the identified mutations. It is our hope that establishment of this form of myopathy accompanied by myogenic tremor as a new disease entity will have important diagnostic and therapeutic implications.

Heterozygous variants in MYBPC1 are associated with an expanded neuromuscular phenotype beyond arthrogryposis.

Encoding the slow skeletal muscle isoform of myosin binding protein-C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M-motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M-motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M-motif to myosin, which likely impairs the formation of actomyosin cross-bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early-onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis.

Unraveling obscurins in heart disease.

Obscurins, expressed from the single OBSCN gene, are a family of giant, modular, cytoskeletal proteins that play key structural and regulatory roles in striated muscles. They were first implicated in the development of heart disease in 2007 when two missense mutations were found in a patient diagnosed with hypertrophic cardiomyopathy (HCM). Since then, the discovery of over a dozen missense, frameshift, and splicing mutations that are linked to various forms of cardiomyopathy, including HCM, dilated cardiomyopathy (DCM), and left ventricular non-compaction (LVNC), has highlighted OBSCN as a potential disease-causing gene. At this time, the functional consequences of the identified mutations remain largely elusive, and much work has yet to be done to characterize the disease mechanisms of pathological OBSCN variants. Herein, we describe the OBSCN mutations known to date, discuss their potential impact on disease development, and provide future directions in order to better understand the involvement of obscurins in heart disease.

Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.

Myosin binding protein-C slow (sMyBP-C) comprises a family of accessory proteins in skeletal muscles that bind both myosin and actin filaments. Herein, we examined the role of sMyBP-C in adult skeletal muscles using in vivo gene transfer and clustered regularly interspaced short palindromic repeats technology to knock down all known sMyBP-C variants. Our findings, confirmed in two different skeletal muscles, demonstrated efficient knockdown (KD) of sMyBP-C (>70%) resulting in notably decreased levels of thick, but not thin, filament proteins ranging from ∼50% for slow and fast myosin to ∼20% for myomesin. Consistent with this, A bands were selectively distorted, and sarcomere length was significantly reduced. Contrary to earlier in vitro studies showing that addition of recombinant sMyBP-C slows down the formation of actomyosin crossbridges, our work demonstrates that KD of sMyBP-C in intact myofibers results in decreased contraction and relaxation kinetics under no-load conditions. Similarly, KD muscles develop markedly reduced twitch and tetanic force and contraction velocity. Taken together, our results show that sMyBP-C is essential for the regular organization and maintenance of myosin filaments into A bands and that its structural role precedes its ability to regulate actomyosin crossbridges.-Geist, J., Ward, C. W., Kontrogianni-Konstantopoulos, A. Structure before function: myosin binding protein-C slow is a structural protein with regulatory properties.

Novel obscurins mediate cardiomyocyte adhesion and size via the PI3K/AKT/mTOR signaling pathway.

The intercalated disc of cardiac muscle embodies a highly-ordered, multifunctional network, essential for the synchronous contraction of the heart. Over 200 known proteins localize to the intercalated disc. The challenge now lies in their characterization as it relates to the coupling of neighboring cells and whole heart function. Using molecular, biochemical and imaging techniques, we characterized for the first time two small obscurin isoforms, obscurin-40 and obscurin-80, which are enriched at distinct locations of the intercalated disc. Both proteins bind specifically and directly to select phospholipids via their pleckstrin homology (PH) domain. Overexpression of either isoform or the PH-domain in cardiomyocytes results in decreased cell adhesion and size via reduced activation of the PI3K/AKT/mTOR pathway that is intimately linked to cardiac hypertrophy. In addition, obscurin-80 and obscurin-40 are significantly reduced in acute (myocardial infarction) and chronic (pressure overload) murine cardiac-stress models underscoring their key role in maintaining cardiac homeostasis. Our novel findings implicate small obscurins in the maintenance of cardiomyocyte size and coupling, and the development of heart failure by antagonizing the PI3K/AKT/mTOR pathway.

Competition through dimerization between antiapoptotic and proapoptotic HS-1-associated protein X-1 (Hax-1).

Studies on Hax-1 have mainly focused on variant (v) 1, demonstrating its antiapoptotic properties. However, HAX1 is heavily spliced, generating structurally distinct isoforms. We sought to characterize the Hax-1 isoforms expressed in rat heart before and after insult. We confirmed the presence of at least four Hax-1 transcripts in healthy rat cardiac muscle. These exhibited differential expression before and after induction of myocardial infarction, with v2 being up-regulated 12-fold at the transcript level and 1.5-fold at the protein level post-insult. Contrary to antiapoptotic rat and human v1, overexpression of rat v2 or human v4 (the human homologue of rat v2) in epithelial cells exacerbated cell death by 30% following H2O2 treatment compared with control vector. Coexpression of rat v1 and v2 or human v1 and v4 neutralized the protective effects of rat and human v1 and the proapoptotic effects of rat v2 and human v4 by modulating cytochrome c release. This is, at least partly, mediated by the ability of Hax-1 proteins to form homotypic and heterotypic dimers with binding affinities ranging from ~3.8 nm for v1 dimers to ~97 nm for v1/v2 dimers. The minimal binding region supporting these interactions lies between amino acids 97-278, which are shared by nearly all Hax-1 proteins, indicating that additional factors regulate the preferential formation of Hax-1 homo- or heterodimers. Our studies are the first to show that Hax-1 is a family of anti- and proapoptotic regulators that may modulate cell survival and death through homo- or heterodimerization.

The kinase domains of obscurin interact with intercellular adhesion proteins.

Obscurins comprise a family of giant (~870- to 600-kDa) and small (~250- to 55-kDa) proteins that play important roles in myofibrillogenesis, cytoskeletal organization, and cell adhesion and are implicated in hypertrophic cardiomyopathy and tumorigenesis. Giant obscurins are composed of tandem structural and signaling motifs, including 2 serine/threonine kinase domains, SK1 and SK2, present at the COOH terminus of giant obscurin-B. Using biochemical and cellular approaches, we show for the first time that both SK1 and SK2 possess enzymatic activities and undergo autophosphorylation. SK2 can phosphorylate the cytoplasmic domain of N-cadherin, a major component of adherens junctions, and SK1 can interact with the extracellular domain of the ß1-subunit of the Na(+)/K(+)-ATPase, which also resides in adherens junctions. Immunostaining of nonpermeabilized myofibers and cardiocytes revealed that some obscurin kinase isoforms localize extracellularly. Quantification of the exofacial expression of obscurin kinase proteins indicated that they occupy ~16 and ~5% of the sarcolemmal surface in myofibers and cardiocytes, respectively. Treatment of heart lysates with peptide-N-glycosidase F revealed that while giant obscurin-B localizes intracellularly, possessing dual kinase activity, a small obscurin kinase isoform that contains SK1 localizes extracellularly, where it undergoes N-glycosylation. Collectively, our studies demonstrate that the obscurin kinase domains are enzymatically active and may be involved in the regulation of cell adhesion.

Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow.

Myosin binding protein C (MyBP-C) is expressed in striated muscles, where it plays key roles in the modulation of actomyosin cross-bridges. Slow MyBP-C (sMyBP-C) consists of multiple variants sharing common domains but also containing unique segments within the NH2 and COOH termini. Two missense mutations in the NH2 terminus (W236R) and COOH terminus (Y856H) of sMyBP-C have been causally linked to the development of distal arthrogryposis-1 (DA-1), a severe skeletal muscle disorder. Using a combination of in vitro binding and motility assays, we show that the COOH terminus mediates binding of sMyBP-C to thick filaments, while the NH2 terminus modulates the formation of actomyosin cross-bridges in a variant-specific manner. Consistent with this, a recombinant NH2-terminal peptide that excludes residues 34-59 reduces the sliding velocity of actin filaments past myosin heads from 9.0 ± 1.3 to 5.7 ± 1.0 µm/s at 0.1 µM, while a recombinant peptide that excludes residues 21-59 fails to do so. Notably, the actomyosin regulatory properties of sMyBP-C are completely abolished by the presence of the DA-1 mutations. In summary, our studies are the first to show that the NH2 and COOH termini of sMyBP-C have distinct functions, which are regulated by differential splicing, and are compromized by the presence of missense point mutations linked to muscle disease.

Compound heterozygous variants in MYBPC1 lead to severe distal arthrogryposis type-1 manifestations.

Dominant missense variants in MYBPC1 encoding slow Myosin Binding Protein-C (sMyBP-C) have been increasingly linked to arthrogryposis syndromes and congenital myopathy with tremor. Herein, we describe novel compound heterozygous variants - NM_002465.4:[c.2486_2492del];[c.2663A > G] - present in fibronectin-III (Fn-III) C7 and immunoglobulin (Ig) C8 domains, respectively, manifesting as severe, early-onset distal arthrogryposis type-1, with the carrier requiring intensive care and several surgical interventions at an early age. Computational modeling predicts that the c.2486_2492del p.(Lys829IlefsTer7) variant destabilizes the structure of the Fn-III C7 domain, while the c.2663A > G p.(Asp888Gly) variant causes minimal structural alterations in the Ig C8 domain. Although the parents of the proband are heterozygous carriers for a single variant, they exhibit no musculoskeletal defects, suggesting a complex interplay between the two mutant alleles underlying this disorder. As emerging novel variants in MYBPC1 are shown to be causatively associated with musculoskeletal disease, it becomes clear that MYBPC1 should be included in relevant genetic screenings.

Essential role of obscurin kinase-1 in cardiomyocyte coupling via N-cadherin phosphorylation.

Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent Kcat of approximately 5.05 min-1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.

Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1.

Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca(2+) channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca(2+) were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.

Obscurins: unassuming giants enter the spotlight.

Discovered about a decade ago, obscurin (~720 kDa) is a member of a family of giant proteins expressed in striated muscle that are essential for normal muscle function. Much of what we understand about obscurin stems from its functions in cardiac and skeletal muscle. However, recent evidence has indicated that variants of obscurin ("obscurins") are expressed in diverse cell types, where they contribute to distinct cellular processes. Dysfunction or abrogation of obscurins has also been implicated in the development of several pathological conditions, including cardiac hypertrophy and cancer. Herein, we present an overview of obscurins with an emphasis on novel findings that demonstrate their heretofore-unsuspected importance in cell signaling and disease progression.

Loss of giant obscurins promotes breast epithelial cell survival through apoptotic resistance.

Obscurins (∼70 - 870 kDa), encoded by the single OBSCN gene, are cytoskeletal proteins originally identified in striated muscles with structural and regulatory roles. Recently, analysis of 13,023 genes in breast and colorectal cancers identified OBSCN as one of the most frequently mutated genes, implicating it in cancer formation. Herein we studied the expression profile of obscurins in breast, colon, and skin cancer cell lines and their involvement in cell survival. Immunoblot analysis demonstrated significant reduction of obscurin proteins [corrected] in cancer cells, resulting from decreased mRNA levels and/or the presence of mutant transcripts. In normal epithelium, obscurins localize in cytoplasmic puncta, the cell membrane, and the nucleus. Accordingly, subcellular fractionation demonstrated the presence of 2 novel nuclear isoforms of ∼110 and ∼120 kDa. Nontumorigenic MCF10A breast epithelial cells stably transduced with shRNAs targeting giant obscurins exhibited increased viability (∼30%) and reduced apoptosis (∼20%) following exposure to the DNA-damaging agent etoposide. Quantitative RT-PCR further indicated that the antiapoptotic genes BAG4 and HAX1 were up-regulated (1.5- and 1.4-fold, respectively), whereas initiator caspase-9 and death caspase-3 transcripts were down-regulated (0.8- and 0.6-fold, respectively). Our findings are the first to pinpoint critical roles for obscurins in cancer development by contributing to the regulation of cell survival.

Alterations in cytoskeletal and Ca2+ cycling regulators in atria lacking the obscurin Ig58/59 module.

Introduction: Obscurin (720-870 kDa) is a giant cytoskeletal and signaling protein that possesses both structural and regulatory functions in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin bind to a diverse set of proteins that are essential for the proper structure and function of the heart, including giant titin, novex-3, and phospholamban (PLN). Importantly, the pathophysiological significance of the Ig58/59 module has been further underscored by the discovery of several mutations within Ig58/59 that are linked to various forms of myopathy in humans. We previously generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59, and characterized the effects of this deletion on cardiac morphology and function through aging. Our findings demonstrated that Obscn-ΔIg58/59 male animals develop severe arrhythmia, primarily manifesting as episodes of junctional escape and spontaneous loss of regular p-waves, reminiscent of human atrial fibrillation, accompanied by significant atrial enlargement that progresses in severity with aging. Methods and Results: To comprehensively characterize the molecular alterations responsible for these pathologies, we performed proteomic and phospho-proteomic analyses in aging Obscn-ΔIg58/59 atria. Our studies revealed extensive and novel alterations in the expression and phosphorylation profile of major cytoskeletal proteins, Ca2+ regulators, and Z-disk associated protein complexes in the Obscn-ΔIg58/59 atria through aging. Discussion: These studies implicate obscurin, particularly the Ig58/59 module, as an essential regulator of the Z-disk associated cytoskeleton and Ca2+ cycling in the atria and provide new molecular insights into the development of atrial fibrillation and remodeling.

The anti-apoptotic protein HAX-1 is a regulator of cardiac function.

The HS-1 associated protein X-1 (HAX-1) is a ubiquitously expressed protein that protects cardiomyocytes from programmed cell death. Here we identify HAX-1 as a regulator of contractility and calcium cycling in the heart. HAX-1 overexpression reduced sarcoplasmic reticulum Ca-ATPase (SERCA2) pump activity in isolated cardiomyocytes and in vivo, leading to depressed myocyte calcium kinetics and mechanics. Conversely, downregulation of HAX-1 enhanced calcium cycling and contractility. The inhibitory effects of HAX-1 were abolished upon phosphorylation of phospholamban, which plays a fundamental role in controlling basal contractility and constitutes a key downstream effector of the beta-adrenergic signaling cascade. Mechanistically, HAX-1 promoted formation of phospholamban monomers, the active/inhibitory units of the calcium pump. Indeed, ablation of PLN rescued HAX-1 inhibition of contractility in vivo. Thus, HAX-1 represents a regulatory mechanism in cardiac calcium cycling and its responses to sympathetic stimulation, implicating its importance in calcium homeostasis and cell survival.

Giant obscurin regulates migration and metastasis via RhoA-dependent cytoskeletal remodeling in pancreatic cancer.

Obscurins, encoded by the OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. Large scale omics analyses reveal that OBSCN is highly mutated across different types of cancer, exhibiting a 5-8% mutation frequency in pancreatic cancer. Yet, the functional role of OBSCN in pancreatic cancer progression and metastasis has to be delineated. We herein show that giant obscurins are highly expressed in normal pancreatic tissues, but their levels are markedly reduced in pancreatic ductal adenocarcinomas. Silencing of giant obscurins in non-tumorigenic Human Pancreatic Ductal Epithelial (HPDE) cells and obscurin-expressing Panc5.04 pancreatic cancer cells induces an elongated, spindle-like morphology and faster cell migration via cytoskeletal remodeling. Specifically, depletion of giant obscurins downregulates RhoA activity, which in turn results in reduced focal adhesion density, increased microtubule growth rate and faster actin dynamics. Although OBSCN knockdown is not sufficient to induce de novo tumorigenesis, it potentiates tumor growth in a subcutaneous implantation model and exacerbates metastasis in a hemispleen murine model of pancreatic cancer metastasis, thereby shortening survival. Collectively, these findings reveal a critical role of giant obscurins as tumor suppressors in normal pancreatic epithelium whose loss of function induces RhoA-dependent cytoskeletal remodeling, and promotes cell migration, tumor growth and metastasis.

NEB mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy.

Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients' muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin-deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin-deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.