Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration

Differential mechanisms for TRIM32-mediated muscle tissue growth and homeostasis

Doctor of PhilosophyBiochemistry and Molecular Biophysics Interdepartmental ProgramErika R. GeisbrechtThe decision of a cell or tissue to undergo growth or maintain homeostasis requires a delicate balance between signaling pathways. In situations that require rapid growth, metabolism may be rewired to increase cell size. One such pathway, termed glycolysis, utilizes glucose to generate energy and metabolic intermediates to produce cellular constituents, including proteins, sugars, and lipids, necessary for building biomass. This is similar to the ‘Warburg effect,’ whereby cancer cells redirect the availability of energetic substrates to generate building blocks required for the uncontrolled growth and proliferation of cells. However, mechanisms that control and promote the utilization of metabolic intermediates for the accumulation of cellular material are not fully understood. The unifying theme of this dissertation is to understand how the E3 ubiquitin ligase TRIM32 mediates the regulation and maintenance of muscle tissue in Drosophila melanogaster, where it is well-established that larval muscles alter their metabolic profile towards glycolysis to allow for a 200-fold increase in growth during development. Previous studies in our lab found that loss of Drosophila TRIM32, orthologous to the gene mutated in patients with Limb-girdle muscular dystrophy type 2H (LGMD2H), exhibit smaller muscles with progressive tissue degeneration. It was assumed that this reduced cell size was a secondary consequence of muscle deterioration. However, we made the surprising discovery that TRIM32 physically interacts with two enzymes that function in glycolysis. Furthermore, loss of TRIM32 reduces glycolytic flux, thus limiting the ability of muscle cells to produce cellular building blocks required for growth. Mutations in TRIM32 also cause a reduction in the overall size of the developing larval brain, another tissue with high glycolytic activity. This ‘Warburg-like’ elevated glycolytic rate that operates in larval muscle and brain tissue is analogous to the altered metabolism in rapidly proliferating cancer or stem cells. Since TRIM32 is upregulated in multiple types of cancer, we hypothesized that TRIM32 could be a general regulator of highly glycolytic tumor cells. Using a Drosophila wing disc tumor model, we found that Pvr-induced epithelial tumors are reduced in size upon removal of TRIM32. These findings suggest that TRIM32 directly controls cell growth, not just in muscle tissue, but in other cell types that exhibit altered metabolism to increase cell size. The pathological alleles present in LGMD2H patients vary in origin, consisting of point mutations (R394H, D487N), a single amino acid deletion (D588Δ), frameshift deletions (A422fs, T520fs, L535fs, I590fs), and a stop codon (R613*). It is not clear how these conserved mutations alter TRIM32 function. We hypothesized that similar to loss of TRIM32, LGMD2H mutations also reduce muscle size, contributing to disease pathogenesis. However, we found that transgenic expression of some of these myopathic alleles (R394H, D487N and 520fs) exhibit normal muscle growth, but rather induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels. These results mimic phenotypes in patients afflicted with LGMD2H. We further uncovered that levels of the transmembrane proteins βPS integrin and Sarcoglycan δ (Scgδ), both core components of costameres, are elevated in Drosophila muscles expressing TRIM32 disease alleles and in mouse C2C12 muscle cells expressing an inactive version of TRIM32. These studies, taken together, have identified two independent roles of TRIM32 in the growth and maintenance of muscle tissue. TRIM32 stabilizes glycolytic enzymes to stimulate muscle growth and likely controls protein turnover of costamere components essential for myofibril stability and integrity.

Exploring the molecular mechanisms of Drosophila dTRIM32 implicated in pathogenesis of Limb-Girdle Muscular Dystrophy 2H

Master of ScienceBiochemistry and Molecular Biophysics Interdepartmental ProgramErika Rae GeisbrechtThe E3 ubiquitin ligase TRIM32 is a member of tripartite motif (TRIM) family of proteins involved in various processes including differentiation, cell growth, muscle regeneration and cancer. TRIM32 is conserved between vertebrates (humans, mouse) and invertebrates (Drosophila). The N-terminus of this protein is characterized by a RING domain, B-box domain, and Coiled-Coil region, while the C-terminus contains six NHL repeats. In humans, mutations that cluster in the NHL domains of TRIM32 result in the muscle disorders Limb-Girdle Muscular Dystrophy type 2H (LGMD2H) and Sarcotubular Myopathy (STM). Mutations in the B-box region cause Bardet-Biedl Syndrome (BBS), a clinically separate disorder that affects multiple parts of the body. A comprehensive genetic analysis in vertebrate models is complicated by the ubiquitous expression of TRIM32 and neurogenic defects in TRIM32-/- mutant mice that are independent of the muscle pathology associated with LGMD2H. The model organism Drosophila melanogaster possesses a TRIM32 [dTRIM32/Thin (Tn)/Abba] homolog highly expressed in muscle tissue. We previously showed that dTRIM32 is localized to Z-disk of the sarcomere and is required for myofibril stability. Muscles form correctly in Drosophila tn mutants, but exhibit a degenerative muscle phenotype once contraction ensues. Mutant or RNAi knockdown larvae are also defective in locomotion, which mimics clinical features associated with loss of TRIM32 in LGMD2H patients. It is predicted that mutations in the NHL domain either affect protein structure or are involved in protein-protein interactions. However, the molecular mechanism by which these mutations affect the interaction properties of dTRIM32 is not understood. Biochemical pulldown assays using the bait fusion protein GST-dTRIM32-NHL identified numerous dTRIM32 binding proteins in larval muscle tissue. Many key glycolytic enzymes were present in the dTRIM32 pulldowns and not in control experiments. Glycolytic genes are expressed in the developing Drosophila musculature and are required for myoblast fusion. Strikingly, many glycolytic proteins are also found at the Z-disk, consistent with dTRIM32 localization. Our biochemical and genetic studies provide evidence that there is direct interaction between dTRIM32 and glycolytic proteins (Aldolase and PGLYM). dTRIM32 also regulates glycolytic enzyme levels and protein localization at their sites of action. These data together suggest a role for dTRIM32 in coordinating glycolytic enzyme function, possibly for localized ATP production or to maintain muscle mass via glycolytic intermediates

TRIM32: A Multifunctional Protein Involved in Muscle Homeostasis, Glucose Metabolism, and Tumorigenesis

Human tripartite motif family of proteins 32 (TRIM32) is a ubiquitous multifunctional protein that has demonstrated roles in differentiation, muscle physiology and regeneration, and tumor suppression. Mutations in TRIM32 result in two clinically diverse diseases. A mutation in the B-box domain gives rise to Bardet–Biedl syndrome (BBS), a disease whose clinical presentation shares no muscle pathology, while mutations in the NHL (NCL-1, HT2A, LIN-41) repeats of TRIM32 causes limb-girdle muscular dystrophy type 2H (LGMD2H). TRIM32 also functions as a tumor suppressor, but paradoxically is overexpressed in certain types of cancer. Recent evidence supports a role for TRIM32 in glycolytic-mediated cell growth, thus providing a possible mechanism for TRIM32 in the accumulation of cellular biomass during regeneration and tumorigenesis, including in vitro and in vivo approaches, to understand the broad spectrum of TRIM32 functions. A special emphasis is placed on the utility of the Drosophila model, a unique system to study glycolysis and anabolic pathways that contribute to the growth and homeostasis of both normal and tumor tissues