The tripartite motif (TRIM) gene family is a highly conserved group of E3 ubiquitin ligase proteins that can establish substrate specificity for the ubiquitin-proteasome complex and also have proteasome-independent functions. While several family members were studied previously, it is relatively recent that over 80 genes, based on sequence homology, were grouped to establish the TRIM gene family. Functional studies of various TRIM genes linked these proteins to modulation of inflammatory responses showing that they can contribute to a wide variety of disease states including cardiovascular, neurological and musculoskeletal diseases, as well as various forms of cancer. Given the fundamental role of the ubiquitin-proteasome complex in protein turnover and the importance of this regulation in most aspects of cellular physiology, it is not surprising that TRIM proteins display a wide spectrum of functions in a variety of cellular processes. This broad range of function and the highly conserved primary amino acid sequence of family members, particularly in the canonical TRIM E3 ubiquitin ligase domain, complicates the development of therapeutics that specifically target these proteins. A more comprehensive understanding of the structure and function of TRIM proteins will help guide therapeutic development for a number of different diseases. This review summarizes the structural organization of TRIM proteins, their domain architecture, common and unique post-translational modifications within the family, and potential binding partners and targets. Further discussion is provided on efforts to target TRIM proteins as therapeutic agents and how our increasing understanding of the nature of TRIM proteins can guide discovery of other therapeutics in the future.
Various previous studies established that the amphiphilic tri-block copolymer known as poloxamer 188 (P188) or Pluronic-F68 can stabilize the plasma membrane following a variety of injuries to multiple mammalian cell types. This characteristic led to proposals for the use of P188 as a therapeutic treatment for various disease states, including muscular dystrophy. Previous studies suggest that P188 increases plasma membrane integrity by resealing plasma membrane disruptions through its affinity for the hydrophobic lipid chains on the lipid bilayer. P188 is one of a large family of copolymers that share the same basic tri-block structure consisting of a middle hydrophobic propylene oxide segment flanked by two hydrophilic ethylene oxide moieties [poly(ethylene oxide)80-poly(propylene oxide)27-poly(ethylene oxide)80]. Despite the similarities of P188 to the other poloxamers in this chemical family, there has been little investigation into the membrane-resealing properties of these other poloxamers. In this study we assessed the resealing properties of poloxamers P181, P124, P182, P234, P108, P407, and P338 on human embryonic kidney 293 (HEK293) cells and isolated muscle from the mdx mouse model of Duchenne muscular dystrophy. Cell membrane injuries from glass bead wounding and multiphoton laser injury show that the majority of poloxamers in our panel improved the plasma membrane resealing of both HEK293 cells and dystrophic muscle fibers. These findings indicate that many tri-block copolymers share characteristics that can increase plasma membrane resealing and that identification of these shared characteristics could help guide design of future therapeutic approaches.
Various injuries to the neural tissues can cause irreversible damage to multiple functions of the nervous system ranging from motor control to cognitive function. The limited treatment options available for patients have led to extensive interest in studying the mechanisms of neuronal regeneration and recovery from injury. Since many neurons are terminally differentiated, by increasing cell survival following injury it may be possible to minimize the impact of these injuries and provide translational potential for treatment of neuronal diseases. While several cell types are known to survive injury through plasma membrane repair mechanisms, there has been little investigation of membrane repair in neurons and even fewer efforts to target membrane repair as a therapy in neurons. Studies from our laboratory group and others demonstrated that mitsugumin 53 (MG53), a muscle-enriched tripartite motif (TRIM) family protein also known as TRIM72, is an essential component of the cell membrane repair machinery in skeletal muscle. Interestingly, recombinant human MG53 (rhMG53) can be applied exogenously to increase membrane repair capacity both in vitro and in vivo. Increasing the membrane repair capacity of neurons could potentially minimize the death of these cells and affect the progression of various neuronal diseases. In this study we assess the therapeutic potential of rhMG53 to increase membrane repair in cultured neurons and in an in vivo mouse model of neurotrauma. We found that a robust repair response exists in various neuronal cells and that rhMG53 can increase neuronal membrane repair both in vitro and in vivo. These findings provide direct evidence of conserved membrane repair responses in neurons and that these repair mechanisms can be targeted as a potential therapeutic approach for neuronal injury.
Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10–20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10–20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.
A common aspect of many diseases and traumatic injuries affecting skeletal muscle is the necrotic death of muscle fibers. Necrotic death of muscle fibers involves the breakdown of the sarcolemmal membrane that can be exacerbated by increased fragility of the membrane or by compromised endogenous sarcolemmal membrane repair processes. Increasing the efficacy of these repair mechanisms could act as a therapeutic approach for several muscular diseases and injuries. Limb‐girdle muscular dystrophy 2B (LGMD2B), a subtype of LGMD, is an autosomal recessive neuromuscular disorder caused by mutations in the DYSF gene that encodes dysferlin, a protein that is vital in membrane repair. The dysferlin protein has been previously shown to facilitate membrane repair in skeletal muscle and knockout mice for dysferlin develop progressive muscular dystrophy. When dysferlin is absent or inactivated through mutation, membrane damage that cannot be repaired accumulates until muscle fibers undergo necrosis that eventually overwhelms the regenerative capacity of the muscle. This causes skeletal muscle deterioration in LGMD2B patients, particularly in the pelvic and shoulder girdle muscles. There is currently no cure for LGMD2B and treatments that stimulate the membrane repair process in muscle fibers have been greatly overlooked and underutilized as a potential therapeutic approach. This project investigates a novel membrane repair signaling cascade in skeletal cells by utilizing SC79, an Akt activator, to increase membrane repair capacity in skeletal muscle. We hypothesize that activation of the phosphoinositide‐3 kinase (PI3K)/Akt1 signaling axis regulates membrane repair in cultured muscle cells and mouse tissue. Based on our data, we find that SC79 injection leads to decreased sarcolemmal membrane injury during treadmill exercises where injection of SC79 decreases the entry of Evans blue dye into muscle fibers. We determined that various doses of SC79 can increase membrane repair in cultured muscle cells and Bla/J dysferlin deficient mice. In these studies, multi‐photon infrared laser microscopy was used to damage the cell membrane of myoblasts, transdifferentiated from skin fibroblasts isolated from LGMD2B patients, in the presence of FM4‐64 dye, a lipophilic dye that fluoresces when it enters the cells and binds to the phospholipids of the cell membrane and intracellular organelles. The extent of localized FM4‐64 dye fluorescence provides a measurement of how well the membrane is repaired. We found SC79 could increase membrane repair in these human patient myoblasts in a dose dependent fashion. This assay was also used on isolated complete muscles from the Bla/J dysferlin deficient mice, where SC79 also increased membrane repair responses. We conclude that activation of the PI3K/Akt1 signaling axis increases membrane repair in dysferlin deficient skeletal muscle. This project explores a novel signaling cascade controlling membrane repair that could be leveraged to develop new therapies for muscle disease and injury.
S135 hand-function PROs have a higher correlation with pinch and grip strength than the IBM-FRS.
Cell membrane repair is an essential cellular process to repair damage to the membrane and maintain the integrity of the cell membrane. Impaired membrane resealing kinetics can potentially lead to neurodegenerative diseases. Enhancing membrane repair via therapeutic intervention could minimize neurodegeneration in neuronal disease states. Recombinant human MG53 (rhMG53) and poloxamers have been an effective therapeutic to enhance membrane repair in muscle and non‐muscle cell types. Here, we aimed to assess if the membrane repair response is active and the efficacy of enhancing membrane resealing in neuronal cells. To assess if neuronal cells have an active membrane repair response, mouse neuroblastoma N2a cells were transfected with GFP or GFP‐MG53. MG53 localization was tracked following multiphoton infrared laser injury. N2a cells were transfected with GFP or GFP‐MG53, and subjected to multiphoton infrared laser injury to investigate if MG53 expression enhances membrane resealing following damage. An infrared laser was used to damage the cell membrane of N2a cells and primary mouse dorsal root ganglion neurons (DRG) in the presence of FM4‐64 dye. To test the efficacy of therapeutics, N2as and DRGs were treated with 1μM rhMG53 or 100μM Poloxamer 188 (P188) and challenged with the multiphoton laser injury. Lastly, the therapeutic potential of rhMG53 was tested in vivo with a sciatic nerve crush injury model. C57Bl/6 mice sciatic nerves were mechanically crushed for 10 seconds and then injected with 1μL rhMG53 (1mg/mL) or saline. The sciatic nerves were harvested and immunostained with an axonal regeneration marker, SCG10. SCG10 staining intensity was measured to calculate the regeneration index. Overall, we observed MG53 trafficking to the injury site which indicates there is an activate membrane repair mechanism within neuronal cells. Therapeutic levels of rhMG53 and P188 increased membrane resealing in both N2as and DRGs. Lastly, rhMG53‐treated sciatic nerves displayed a two‐fold increase in regenerative capacity following sciatic nerve crush injury compared to the saline vehicle control. These results illustrate an active membrane repair process in neuronal cells and potential for therapeutic intervention in the context of neuronal diseases, specifically neurodegenerative diseases.
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