Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments.     

Μyospryn: a multifunctional desmin-associated protein

Desmin, the muscle-specific intermediate filament protein, forms a 3D scaffold that links the contractile apparatus to the costameres of plasma membrane, intercalated disks, the nucleus, and also other membranous organelles. The cellular scaffold formed by desmin and its binding partners might be implicated in signaling and trafficking processes, vital mechanisms for the survival of the mammalian cell. One novel desmin-associated protein is the tripartite motif-like protein myospryn. Myospryn was initially identified as an associated partner to the biogenesis of lysosome-related organelles complex 1 protein dysbindin, implicating its potential involvement in vesicle trafficking and organelle biogenesis and/or positioning. Myospryn is also an A kinase anchoring protein, raising the possibility that together with desmin and other cytoskeletal and signaling proteins, it could participate in the subcellular targeting of protein kinase A activity in striated muscle. As with desmin, different members of this scaffold might play a crucial role in the pathogenesis of muscle disease, since any disturbance in these highly coordinated signaling pathways is expected to compromise efficient maintenance of structure–function integrity of muscle and lead to different cardiac and skeletal myopathies.     

The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?

Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture.     

A Myopathy-linked Desmin Mutation Perturbs Striated Muscle Actin Filament Architecture

Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.     

Lysine methylation: beyond histones

Posttranslational modifications (PTMs) of histone proteins, such as acetylation, methylation, phosphorylation, and ubiquitylation, play essential roles in regulating chromatin dynamics. Combinations of different modifications on the histone proteins, termed 'histone code' in many cases, extend the information potential of the genetic code by regulating DNA at the epigenetic level. Many PTMs occur on non-histone proteins as well as histones, regulating protein-protein interactions, stability, localization, and/or enzymatic activities of proteins involved in diverse cellular processes. Although protein phosphorylation, ubiquitylation, and acetylation have been extensively studied, only a few proteins other than histones have been reported that can be modified by lysine methylation. This review summarizes the current progress on lysine methylation of non-histone proteins, and we propose that lysine methylation, like phosphorylation and acetylation, is a common PTM that regulates proteins in diverse cellular processes.     

组蛋白泛素化修饰及其在DNA损伤应答中的作用

泛素化修饰是真核生物细胞内重要的翻译后修饰类型,通过调节蛋白质活性、稳定性和亚细胞定位广泛参与细胞内各项信号传导与代谢过程,对维持正常生命活动具有重要意义。组蛋白作为染色质中主要的蛋白成分,与DNA复制转录、修复等行为密切相关,是研究翻译后修饰的热点。DNA损伤后,组蛋白泛素化修饰通过调节核小体结构、激活细胞周期检查点、影响修复因子的招募与装配等诸多途径参与损伤应答。同时,组蛋白泛素化修饰还能调节其他位点翻译后修饰,并通过这种串扰(crosstalk)作用调节DNA损伤应答。本文介绍了组蛋白泛素化修饰的主要位点和相关组分(包括E3连接酶、去泛素化酶与效应分子),以及这些修饰作用共同编译形成的信号网络在DNA损伤应答中的作用,最后总结了目前该领域研究所面临的一些问题,以期为科研人员进一步探索组蛋白密码在DNA损伤应答中的作用提供参考。     

Desmin mutations result in mitochondrial dysfunction regardless of their aggregation properties

Desmin, being a major intermediate filament of muscle cells, contributes to stabilization and positioning of mitochondria. Desmin mutations have been reported in conjunction with skeletal myopathies accompanied by mitochondrial dysfunction. Depending on the ability to promote intracellular aggregates formation, mutations can be considered aggregate-prone or non-aggregate-prone. The aim of the present study was to describe how expression of different desmin mutant isoforms effects mitochondria and contributes to the development of myocyte dysfunction. To achieve this goal, two non-aggregate-prone (Des S12F and Des A213V) and four aggregate-prone (Des L345P, Des A357P, Des L370P, Des D399Y) desmin mutations were expressed in skeletal muscle cells. We showed that all evaluated mutations affected the morphology of mitochondrial network, suppressed parameters of mitochondrial respiration, diminished mitochondrial membrane potential, increased ADP/ATP ratio, and enhanced mitochondrial DNA (mtDNA) release. mtDNA was partially secreted through exosomes as demonstrated by GW4869 treatment. Dysfunction of mitochondria was observed regardless the type of mutation: aggregate-prone or non-aggregate-prone. However, expression of aggregate-prone mutations resulted in more prominent phenotype. Thus, in this comparative study of six pathogenic desmin mutations that cause skeletal myopathy development, we confirmed a role of mitochondrial dysfunction and mtDNA release in the pathogenesis of desmin myopathies, regardless of the aggregation capacity of the mutated desmin.     

Desmin aggregate formation by R120G alphaB-crystallin is caused by altered filament interactions and is dependent upon network status in cells

The R120G mutation in alphaB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of alphaB-crystallin for desmin filaments. The apparent dissociation constant of R120G alphaB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G alphaB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type alphaB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G alphaB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in alphaB-crystallin.     

Assembly of carboxy-terminally deleted desmin in vimentin-free cells.

Using a vimentin-free expression system we were able to demonstrate that the carboxy terminus of desmin is necessary for filament assembly in the living cell. Desmin subunits missing only 4 carboxy-terminal residues of their rod domain are incapable of homopolymeric filament assembly. Moreover, even single amino acid substitutions in the conserved carboxy-terminal part of the rod domain prevent desmin subunits from homopolymeric filament assembly. Desmin subunits missing 18 or more carboxy-terminal residues of their rod domain (including the complete conserved carboxy-terminal region) are unstable in cells devoid of intact type III intermediate filaments (IFs). Interaction with an intact type III IF, however, stabilizes these mutated desmin subunits. Expression of a desmin subunit missing both its non-helical end domains in vimentin-containing cells disrupts the endogenous vimentin network completely.     

Two Desmin Gene Mutations Associated with Myofibrillar Myopathies in Polish Families

Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (DES) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two DES mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on DES expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.     

Intermediate filament reconstitution in vitro. The role of phosphorylation on the assembly-disassembly of desmin

Desmin, the myogenic intermediate filament protein, is a phosphoprotein containing phosphoserine, in vivo. The role of phosphorylation on assembly-disassembly and organization of the desmin filament has remained obscure. We report here on a stable and purified system which enables a biochemical examination of desmin filament assembly and disassembly. Using this in vitro system, we carried out stoichiometrical phosphorylations by purified protein kinases. The extent of polymerization-depolymerization was estimated using procedures related to centrifugation and electron microscopy. The evidence we obtained suggests that disassembly of the desmin filament and inhibition of the NaCl-dependent polymerization of the soluble desmin can reversibly occur with either cAMP-dependent or Ca2+-activated, phospholipid-dependent desmin phosphorylation.     

Structure and elasticity of desmin protofibrils explored with scanning force microscopy

Desmin filaments form the intermediate filament system of muscle cells where they play important role in maintaining mechanical integrity and elasticity. Although the importance of desmin elasticity and assembly-disassembly dynamics in cellular mechanics is being increasingly recognized, the molecular basis of neither desmin's elasticity nor its disassembly pathway is well understood. In the present work, we explored the topographical structure of purified and reconstituted desmin filaments by using scanning force microscopy. With the addition of divalent cation chelators ethyleneglycoltetraacetic acid or ethylenediaminetetraacetic acid, the filaments disassembled on a time scale of hours to days into stable, thin fibrillar components with variable (up to micrometer) length, smooth surface and uniform thickness, which are identified as protofibrils. Desmin protofibrils appear as elastic structures with a persistence length of 51.5 nm, and their Young's modulus (10.6 MPa) far exceeds that of the mature filament (3.7 MPa). Protofibrillar bundling within the desmin filament results in high longitudinal tensile strength at a large bending flexibility. The stability of protofibrils suggests that desmin may disassemble along a pathway quite distinct from its assembly.     

Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex.

We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with alpha-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies.     

Posttranslational modifications of CENP-A: marks of distinction

Centromeres are specialized chromosome domain that serve as the site for kinetochore assembly and microtubule attachment during cell division, to ensure proper segregation of chromosomes. In higher eukaryotes, the identity of active centromeres is marked by the presence of CENP-A (centromeric protein-A), a histone H3 variant. CENP-A forms a centromere-specific nucleosome that acts as a foundation for centromere assembly and function. The posttranslational modification (PTM) of histone proteins is a major mechanism regulating the function of chromatin. While a few CENP-A site-specific modifications are shared with histone H3, the majority are specific to CENP-A-containing nucleosomes, indicating that modification of these residues contribute to centromere-specific function. CENP-A undergoes posttranslational modifications including phosphorylation, acetylation, methylation, and ubiquitylation. Work from many laboratories have uncovered the importance of these CENP-A modifications in its deposition at centromeres, protein stability, and recruitment of the CCAN (constitutive centromere-associated network). Here, we discuss the PTMs of CENP-A and their biological relevance.     

Association between the muscle-specific proteins desmin and caveolin-3 in muscle cells

The muscle-specific intermediate filament protein desmin is expressed in mononucleated myoblasts and in differentiated myotubes. Desmin has been shown to associate with the sarcolemma in specific structures, such as neuromuscular junctions and the dystrophin-associated protein complex. Since these are specialized membrane regions, the study of a possible association between desmin and liquid-ordered membrane microdomains is of particular interest. We have carried out an analysis of the association between desmin and the muscle-specific protein caveolin-3, a major component of caveolar microdomains. Our results demonstrate that (1) desmin precisely co-localizes with caveolin-3 in myoblasts and multinucleated myotubes, (2) caveolin-3 is up-regulated during in vitro chick muscle development, (3) desmin is detectable in caveolae-enriched membrane fractions prepared from skeletal muscle, and (4) caveolin-3 co-immunoprecipitates with desmin. We have thus shown, for the first time, an association between the intermediate filament protein desmin and caveolin-3 in myogenic cells.     

Site-specific antibodies block kinase A phosphorylation of desmin in vitro and inhibit incorporation of myoblasts into myotubes.

Desmin and vimentin are two type III intermediate filament (IF) proteins, which can be phosphorylated in vitro by cAMP-dependent kinase (kinase A) and protein kinase C, and the in vitro phosphorylation of these proteins appears to favor the disassembled state. The sites of phosphorylation for desmin and vimentin have been mapped to their amino-terminal headpiece domains; in chicken smooth muscle desmin the most kinase A-reactive residues are ser-29 and ser-35. In this study we have examined the phosphorylation of desmin by the catalytic subunit of kinase A by using anti-peptide antibodies directed against residues 26-36. The antibodies, which we call anti-D26, recognize both native and denatured desmin and can discriminate between intact desmin and those derivatives that do not possess residues 26-36. Pre-incubation of desmin with affinity purified anti-D26 blocks total kinase A catalyzed incorporation of 32P into desmin by 75-80%. When antibody-treated IFs are subjected to phosphorylation, no filament break-down is observed after 3 hours. Thus anti-D26 antibodies block phosphorylation of IF in vitro. We have also explored the role of desmin phosphorylation in skeletal muscle cell differentiation using these antibodies. Quail embryo cells, induced to differentiate along the myogenic pathway by infection with avian SKV retroviruses expressing the ski oncogene, were microinjected with affinity purified anti-D26 at the mononucleated, myoblast stage. By 24 h post-injection, the vast majority of uninjected cells had fused into multinucleated myotubes, but all microinjected cells were arrested in the process of incorporating into myotubes and remained mononucleated. This observation suggests that kinase A phosphorylation-induced dynamic behavior of the desmin/vimentin IF cytoskeleton may be one of the many cytoskeletal restructuring events that must take place during myoblast fusion.     

Systematic functional prioritization of protein posttranslational modifications

Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.