InteractiveFly: GeneBrief

thin: Biological Overview | References

All growth and/or proliferation may require the reprogramming of metabolic pathways, whereby a switch from oxidative to glycolytic metabolism diverts glycolytic intermediates towards anabolic pathways. This study identified a novel role for TRIM32 in the maintenance of glycolytic flux mediated by biochemical interactions with the glycolytic enzymes Aldolase and Phosphoglycerate mutase. Loss of Drosophila TRIM32, encoded by thin (tn), shows reduced levels of glycolytic intermediates and amino acids. This altered metabolic profile correlates with a reduction in the size of glycolytic larval muscle and brain tissue. Consistent with a role for metabolic intermediates in glycolysis-driven biomass production, dietary amino acid supplementation in tn mutants improves muscle mass. Remarkably, TRIM32 is also required for ectopic growth: loss of TRIM32 in a wing disc-associated tumor model reduces glycolytic metabolism and restricts growth. Overall, these results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth (Biwa, 2020a).

The metabolism of all cells must adapt to meet the energetic and biosynthetic needs of growth and homeostasis. For example, tissues composed of non-dividing, differentiated cells must strike a balance between catabolic pathways that provide energy for cellular homeostasis and anabolic pathways that repair the cell and generate cell-type-specific molecules. In contrast, the metabolic requirements of cell growth and proliferation often require a shift toward anabolic pathways that favors the synthesis of macromolecules, such as proteins, lipids, nucleic acids, and complex carbohydrates. Striking this delicate balance between degradative and biosynthetic processes requires the integration of extracellular and intracellular information by complex signaling networks (Biwa, 2020a).

The mechanisms by which cell proliferation and tissue growth rewire metabolism to enhance biosynthesis are diverse and complex. These changes in metabolic flux involve pathways such as the pyrimidine and purine biosynthesis, one carbon metabolism, and the interplay between the citric acid cycle and amino acids pools. However, the pathway most commonly associated with enhanced biosynthesis is glycolysis, where in biological systems ranging from yeast to human T-cells, glycolytic flux is often elevated in the context of cell growth and proliferation (Zhu, 2019). This observation is particularly apparent in the fruit fly Drosophila melanogaster, where the onset of larval development is preceded by a metabolic switch that induces the coordinate upregulation of genes involved in glycolysis, the pentose phosphate pathway, and lactate dehydrogenase (LDH) (Tennessen, 2011). The resulting metabolic program allows larvae to use dietary carbohydrates for both energy production and biomass accumulation. Moreover, studies of Drosophila larval muscles reveal that this metabolic transition is essential for muscle growth and development, suggesting that glycolysis serves a key role in controlling growth (Tennessen, 2014). The mechanisms that control glycolysis specifically in larval muscle, however, remain relatively unexplored. As a result, Drosophila larval development provides an excellent model for understanding how glycolysis and biomass production are regulated in a rapidly growing tissue. Moreover, since larval muscle increases in size without cell divisions, larval muscle provides an unusual opportunity to understand how glycolytic metabolism promotes growth independent of cell division (Biwa, 2020a).

Of the known factors that promote muscle development, TRIM32 is an intriguing candidate for coordinating metabolism with cell growth. This protein is a member of the Tripartite motif (TRIM)-containing family of proteins defined by an N-terminal RING domain, one or two B-boxes, a coiled-coil domain, and a variable C-terminal region. In TRIM32, six Ncl-1, HT2A, Lin-41 (NHL) repeats comprise the C-terminus and are proposed to mediate the diverse functions of TRIM32, including cell proliferation, neuronal differentiation, muscle physiology and regeneration, and tumorigenesis (Lazzari, 2016; Tocchini, 2015; Watanabe, 2017). A single mutation in the B-box region of TRIM32 causes the multisystemic disorder Bardet-Biedl syndrome (BBS) (Chiang, 2006), while multiple mutations that cluster in the NHL domains result in the muscle disorders Limb-girdle muscular dystrophy type 2H (LGMD2H) and Sarcotubular Myopathy (STM) (Biwa, 2020a).

A complete understanding of TRIM32 function is confounded by its ubiquitous expression and multitude of potential substrates for E3 ligase activity via the RING domain. Many known TRIM32 target substrates include proteins implicated in muscle physiology, consistent with a role for TRIM32 in LGMD2H. However, additional polyubiquitinated substrates, including p53, Abi2, Piasy, XIAP, and MYCN, are implicated in tumorigenesis. Importantly, TRIM32 protein levels are upregulated in multiple tumor types, suggesting that TRIM32 is a key player in growth regulation. There is precedence for NHL function in controlling cell proliferation as two other Drosophila NHL-containing proteins, Brat and Mei-P26, act as tumor suppressors in the larval brain and female germline, respectively (Biwa, 2020a).

This study provide a novel mechanism for TRIM32 in cell growth. The data show that TRIM32 promotes glucose metabolism through the stabilization of glycolytic enzyme levels. This increased rate of TRIM32-mediated glycolytic flux generates precursors that are utilized for biomass production. Surprisingly, this mechanism operates in both non-dividing muscle cells as well as in proliferating larval brain cells, demonstrating a universal metabolic function for TRIM32 in growth control (Biwa, 2020a).

A unique feature of Drosophila larval development is the inherent glycolytic nature of muscle and brain tissue (Li, 2017; Tennessen, 2014; Tixier, 2013), which promotes biomass synthesis during this stage of rapid organismal growth. Maintenance of such a high metabolic rate predicts that enzymes are present at sufficient concentrations in the cell to mediate the rapid shunting of intermediates through the pathway. This study shows that TRIM32 directly interacts with and maintains the levels of two glycolytic enzymes. Decreased protein levels of both Ald and Pglym (and possibly other glycolytic enzymes) cripple this rapid flux, effectively blunting the generation of metabolic intermediates that contribute to anabolic synthesis necessary to sustain cell growth (Biwa, 2020a).

It has been recently reported that pathways controlling lactate and glycerol-3-phosphate metabolism function redundantly in larval growth (Li, 2019). Removal of LDH and hence lactate production caused an increase in glycerol-3-phosphate, which was sufficient to maintain larval redox balance. Since both LDH and GPDH1 regulate redox balance necessary for maintaining high glycolytic flux to promote biomass accumulation, Ldh, Gpdh1 double mutants exhibit severe growth defects with reduced brain size. The current results show that loss of TRIM32 decreases both lactate and glycerol-3-phosphate levels, thus mimicking the reduced carbohydrate metabolism in Ldh, Gpdh1 double mutants (Biwa, 2020a).

It is not clear how mutations in a ubiquitously expressed protein such as TRIM32 result in tissue-specific diseases. One prediction is that TRIM32 differentially interacts with proteins in diverse cell types to elicit distinct biological outputs. There is strong evidence to support this hypothesis in the context of LGMD2H. TRIM32 is upregulated in proliferating satellite cells and loss of this protein prevents myotube regeneration, partially through the misregulation of NDRG and c-Myc. Muscle-specific targets that contribute to disease progression are less clear. TRIM32-mediated deregulation of key muscle substrates, including actin, α-actinin, tropomyosin, and desmin, contribute to muscle atrophy, but studies have not been performed to directly test this model in the context of LGMD2H. Interestingly, mammalian glycolytic type II fibers are preferentially affected over oxidative type I fibers in muscle atrophy induced by aging/starvation, as well as in Duchenne's and Becker muscular dystrophies (DMD). TRIM32 KO muscles also show a decrease in the glycolytic proteins GAPDH and PyK, just as this study observed a reduction in Ald and Pglym levels in tn-/- muscles, suggesting that TRIM32-mediated regulation of glycolysis may be a general mechanism that underlies some muscular dystrophies (Biwa, 2020a).

The multi-faceted roles exhibited by TRIM32 in muscle physiology and cancer seem quite different on the surface. However, control of glycolytic flux may be a common mode of regulation that has been overlooked. As in muscle tissue, the majority of studies on TRIM32 and cancer have focused on identifying substrates that are subject to poly-ubiquitination and subsequent proteasomal degradation. Piasy, p53 and Abi2 are known targets of TRIM32 E3 activity that regulate the proliferative balance in cancer cells (Albor, 2006; Kano, 2008; Liu, 2014). The proteolytic turnover of these proteins may affect signaling pathways independent of glycolytic TRIM32 regulation or may be a compensatory mechanism in response to metabolic shifts in which normal cells can transiently adopt cancer-like metabolism during periods of rapid proliferation (Biwa, 2020a).

How does this loss of TRIM32 lead to a reduction in glycolytic enzymes? Glycolytic proteins may be substrates for TRIM32 E3 ligase activity. It seems unlikely that TRIM32 polyubiquitinates Ald or Pglym for proteasomal degradation as protein levels are not elevated upon loss of this putative E3 activity. Furthermore, co-immunoprecipitaion of higher molecular weight forms of Ald or Pglym with TRIM32 suggests a yet unidentified post-translational modification. Another possibility, which this study favors, is that the NHL domain of TRIM32 serves as a scaffold for the subcellular localization of glycolytic proteins to limit diffusion of substrates during glycolysis. This does not negate, but rather expands the repertoire of TRIM32 functions (Biwa, 2020a).

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. Previous work established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. This study shows 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, 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. It is speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration (Biwa, 2020b).

It is unclear how loss of the single TRIM32 protein in muscle tissue initiates or promotes LGMD2H pathology. Interpretation of mouse TRIM32 models created to mimic LGMD2H pathology has proven complex, as both neurogenic and myopathic abnormalities are present in TRIM32KO or TRIM32 D489N knock-in mice. Moreover, loss of TRIM32 in satellite cells limits muscle regeneration, thereby promoting further tissue deterioration. This stud took advantage of the lack of satellite cells in Drosophila larval muscles to separate the muscle-intrinsic function of TRIM32 from its role in mammalian muscle regeneration. It was confirmed that a decrease in neuronal TRIM32 does not contribute to muscle pathology, while the expression of myopathic mutations in larval and adult muscle leads to pathogenic defects with reduced TRIM32 protein levels. Most importantly, this study has substantiated a role for costameric proteins in disease progression (Biwa, 2020b).

TRIM32 is a multidomain protein that consists of RING domains and NHL repeats that are structurally and functionally conserved among different species. At least 12 different molecules, consisting of either RNA or protein, have been shown to interact with NHL-containing proteins. Moreover, mutations and/or deletions in the NHL region of TRIM family members are linked to diverse human diseases. In addition to LGMD2H caused by mutations in TRIM32, axonal neuropathy and gliomas result from mutations in TRIM2 and TRIM3, respectively. However, understanding of how these repeats function in maintaining adequate protein-protein interactions, especially in muscle cytostructure, is limited (Biwa, 2020b).

Despite the moderate amino acid identity between Drosophila and human TRIM32, this study has demonstrated that structural homology of the entire NHL region is remarkably close and serves as an ideal model to investigate the downstream consequences of these myopathic mutations. Previous modeling of the R394H and D487N mutations used the crystal structure of the related NHL-containing protein Drosophila Brat as a template. The curren domain mutation analysis, based upon the structure solved for TRIM32, reveals alterations that impact not only local protein structure. For example, the R394H mutation located in NHL domain 1 causes backbone perturbations and residue energy changes in NHL domains 3 and 4, thus providing a molecular understanding for the destabilization of the R394H mutant relative to WT NHL by DSC. These results provide evidence that mutations exert debilitating effects on NHL structure and reduce protein levels, further contributing to disease progression (Biwa, 2020b).

Similar to LGMD2H patients, the expression of disease-causing alleles (R394H, D487N and 520fs) in Drosophila larval muscle causes muscle degeneration with reduced TRIM32 protein levels and locomotor defects. It has been reported that RNAi-mediated knockdown of TRIM32 disrupts adult myofiber architecture at day 7 and day 11 after tn RNAi induction posteclosion. In contrast, the current genetic assays show that expression of the R394H and 520fs mutations in IFMs cause severe damage to the myofibers by day 1, which continue to degenerate with age. Expression of the D487N mutation also triggered muscle deterioration, but delayed compared with other mutations. It is also clear that TRIM32 protein levels correlate with protein destabilization and muscle degeneration. TRIM32 did not localize in the IFMs of R394H and 520fs mutants, but remained relatively well localized in D487N mutant myofibers until day 3. In all destabilizing mutations, variable accumulation of TRIM32 puncta was observed. The presence of puncta, rather than the complete absence of protein, may indicate that TRIM32 no longer retains its correct sarcomeric association or possibly represents aggregated TRIM32 protein in response to the damage caused by the myopathic mutations. It is believed that the differences in muscle degeneration and TRIM32 expression in different alleles partially explains the variable onset and phenotype severity observed in LGMD2H patients (Biwa, 2020b).

Do components of the dystrophin-glycoprotein components (DGC) and integrin adhesion system play a critical role in LGMD2H disease progression? Mutations in the DGC and integrin adhesion complex are associated with muscular dystrophies and cardiomyopathies. Deficiency of δ-sarcoglycan in mammalian skeletal muscle results in the absence of α-, β-, and γ-sarcoglycan, suggesting that sarcoglycan is a core component of sarcoglycan complex assembly. Similarly, integrin adhesion molecules at the costamere are indispensable for the development and maintenance of sarcomeric architecture. Work performed in mice and flies showed that integrins play an important role in Z-disk formation. Previous work revealed that TRIM32 is essential for costamere integrity, whereby βPS integrin, talin, spectrin, vinculin, and Scgδ accumulate abnormally along the sarcolemma (LaBeau-DiMenna, 2012). The current results further support that loss of costamere stability may be involved in disease pathogenesis, as the expression of TRIM32 mutations also phenocopies the mislocalization of βPS integrin and Scgδ. Similar data were obtained upon expression of a catalytically inactive version of TRIM32 in murine myoblasts, extending in mammals the novel role of TRIM32 in promoting the degradation of Drosophila costamere components. Importantly, although TM protein is mislocalized upon loss of TRIM32, it retains its normal localization when pathogenic alleles are expressed in muscle tissue. These results suggest that TRIM32 can regulate the levels of sarcomeric proteins such as TM, but this accumulation is not sufficient to cause muscle degeneration as observed with costamere proteins (Biwa, 2020b).

Several studies have shown that upregulation of utrophin or integrin α7 partially compensates for the lack of dystrophin in mdx mice or in human DMD patients. Although the integrin adhesion complex (assayed by βPS integrin) and the DGC complex (assayed by Scgδ) are also up-regulated upon loss of TRIM32, this buildup of costameric proteins at the sarcolemma compromises the attachment between the membrane and myofibrils, leading to muscle degeneration. Some or all of these costamere proteins may be substrates for TRIM32 E3 ligase activity. The abnormal accumulation of βPS integrin and Scgδ along the sarcolemma may result from the inability to be ubiquitinated and turned over by the proteasome and/or the inability to maintain its normal localization due to protein damage during muscle contraction (Biwa, 2020b).

There are conflicting data about whether LGMD2H mutations in the NHL domain of TRIM32 inhibit multimer formation or alter ubiquitination activity. It is speculated that disease-causing mutations elevate protein levels of at least a subset of costamere proteins, either by altering protein interactions or by abolishing the catalytic activity of TRIM32. However, additional work is required to characterize the ubiquitination signature by TRIM32 and the resulting fate of substrate proteins. The structural and functional conservation of TRIM32, combined with the muscle-intrinsic Drosophila genetic model, will continue to provide novel insights into LGMD2H initiation and progression not currently available through study in other model organisms (Biwa, 2020b).

Thin is required for cell death in the Drosophila abdominal muscles by targeting DIAP1

In holometabolous insects, developmentally controlled programmed cell death (PCD) is a conserved process that destroys a subset of larval tissues for the eventual creation of new adult structures. This process of histolysis is relatively well studied in salivary gland and midgut tissues, while knowledge concerning larval muscle destruction is limited. This study examined the histolysis of a group of Drosophila larval abdominal muscles called the dorsal external oblique muscles (DEOMs). Previous studies have defined apoptosis as the primary mediator of DEOM breakdown, whose timing is controlled by ecdysone signaling. However, very little is known about other factors that contribute to DEOM destruction. In this paper, the role of thin (tn), which encodes for the Drosophila homolog of mammalian TRIM32, was examined in the regulation of DEOM histolysis. Loss of Tn blocks DEOM degradation independent of ecdysone signaling. Instead, tn genetically functions in a pathway with the death-associated inhibitor of apoptosis (DIAP1), Dronc, and death-associated APAF1-related killer (Dark) to regulate apoptosis. Importantly, blocking Tn results in the absence of active Caspase-3 immunostaining, upregulation of DIAP1 protein levels, and inhibition of Dronc activation. DIAP1 and Dronc mRNA levels are not altered in tn mutants, showing that Tn acts post-transcriptionally on DIAP1 to regulate apoptosis. This study also found that the RING domain of Tn is required for DEOM histolysis as loss of this domain results in higher DIAP1 levels. Together, these results suggest that the direct control of DIAP1 levels, likely through the E3 ubiquitin ligase activity of Tn, provides a mechanism to regulate caspase activity and to facilitate muscle cell death (Vishal, 2018).

PCD is required for the destruction of certain larval tissues during metamorphosis. Zirin (2013) established that histolysis of the abdominal muscles is regulated by apoptosis, while blocking autophagy does not affect muscle breakdown. In addition to the ecdysone receptor, only a handful of nuclear proteins are known to function in dorsal exterior oblique muscle (DEOM) histolysis. Loss of East results in a partial block in DEOM degeneration, whereas premature muscle destruction is observed in muscles that lack Chromator. Moreover, the two nuclear receptors, FTZ-F1 and HR39, antagonistically function to regulate the timing of DEOM histolysis. This study has further identified Tn as a novel protein in pupal muscle remodeling. However, loss of Tn does not affect salivary gland and midgut histolysis, highlighting an exclusive muscle role for Tn during Drosophila metamorphosis (Vishal, 2018).

Genetic assays demonstrate that tn functions with core components of the cell death machinery to regulate DEOM destruction. It was surprising that inhibition of apoptotic activity in Dark or Dronc mutants was not sufficient to completely block histolysis by 24 h APF. One explanation is the existence of additional cell death mechanisms other than apoptosis. While histolysing DEOMs contained autophagic vesicles, a reduction in autophagy components did not block or delay muscle degradation at 8 h APF. Tests were performed to see if a decrease in Tn-mediated apoptosis could sensitize muscle cells to initiate autophagy as a compensatory mechanism to assure cell death. However, this does not seem to be the case as RNAi knockdown of Atg1, Atg5, or Atg18 does not further block DEOM histolysis in a tn RNAi background. A second explanation is that the hypomorphic nature of these alleles may not completely abrogate Dark and Dronc function. Alternatively, additional effector caspases, including Dcp-1, Decay, and/or Damm, may be operating in the latter stages of DEOM histolysis since these caspases may function redundantly or act independent of the DIAP1-Drice axis (Vishal, 2018).

More than a partial block in DEOM histolysis was expected upon manipulation of DIAP1 (i.e., DIAP1 OE alone or tn RNAi+DIAP1 OE) at 12 h APF. It is possible that normal or overexpressed DIAP1 levels in the DEOMs are not high enough to block apoptosis, especially using RNAi approaches to reduce Tn levels. However, the use of tn-null alleles clearly shows a complete block in muscle degradation and a corresponding inhibition of active Dronc. Seemingly a delicate balance exists to regulate mRNA and protein expression, as well as protein turnover and proteolytic processing of active caspases. Cells must normally prevent cell death and only activate the apoptotic cascade upon a commitment to die. Thus, threshold levels of caspase activity must be reached for this terminal fate. There is evidence for stage or tissue-specific differential sensitivity to pro-apoptotic factors. Early L3 individuals are resistant to apoptosis, while wandering L3 larvae have elevated levels of Dark, Dronc, and Drice that are sufficient to trigger cell death under the appropriate stimuli. A model is proposed whereby Tn, through its RING domain, normally ubiquitinates DIAP1 for delivery to the proteasome during DEOM histolysis. This degradation of DIAP1 relieves Dronc inhibition, thereby initiating the caspase cascade for the execution of cell death. A general reduction in Tn, or loss of RING domain activity, prevents the addition of ubiquitin moieties and causes an increase in DIAP1 levels, effectively blocking cell death by limiting caspase activity (Vishal, 2018).

Numerous roles have been identified for mammalian TRIM32 in normal and cancerous cells. In muscle, mutations in the NHL repeats result in limb-girdle muscular dystrophy type 2H or sarcotubular myopathy. Several structural muscle proteins are targets of TRIM32 activity, including tropomyosin, desmin, α-actinin, and dysbindin. However, it is not yet clear if regulation of these muscle substrates contributes to normal muscle physiology, is required to prevent atrophy, or plays a critical role in disease pathology. The TRIM32-mediated degradation of additional protein substrates, including p53, Abi2, Piasy, and the X-linked IAP (XIAP), contribute to oncogenic or tumor suppressor activities that either confer resistance or susceptibility to apoptosis. Tumor necrosis factor-α can trigger death receptor-mediated apoptosis through the regulation of XIAP activity. TRIM32 colocalizes and directly interacts with XIAP in human kidney epithelial cells (HEK293). Moreover, TRIM32 induces apoptosis through the direct ubiquitination and subsequent protein turnover of XIAP degradation (Ryu, 2011). This control of apoptotic cell death mirrors the current genetic results, strongly suggesting that this TRIM32-mediated regulation of IAP family members may be a conserved mechanism to regulate apoptosis. It would be interesting to further investigate if Tn and mammalian TRIM32 regulates apoptotic decisions in other contexts of muscle development and/or disease (Vishal, 2018).

Thin, a Trim32 ortholog, is essential for myofibril stability and is required for the integrity of the costamere in Drosophila

Myofibril stability is required for normal muscle function and maintenance. Mutations that disrupt myofibril stability result in individuals who develop progressive muscle wasting, or muscular dystrophy, and premature mortality. This study presents investigations of the Drosophila l(2)thin [l(2)tn] mutant. The 'thin' phenotype exhibits features of the human muscular disease phenotype in that tn mutant larvae show progressive muscular degeneration. Loss-of-function and rescue experiments determined that l(2)tn is allelic to the tn locus. tn encodes a TRIM (tripartite motif) containing protein highly expressed in skeletal muscle and is orthologous to the human limb-girdle muscular dystrophy type 2H disease gene Trim32. Thin protein is localized at the Z-disk in muscle, but l(2)tn mutants showed no genetic interaction with mutants affecting the Z-line-associated protein muscle LIM protein 84B. l(2)tn, along with loss-of-function mutants generated for tn, showed no relative mislocalization of the Z-disk proteins alpha-Actinin and muscle LIM protein 84B. In contrast, tn mutants had significant disorganization of the costameric orthologs beta-integrin, Spectrin, Talin, and Vinculin, and the initial description is presented for the costamere, a key muscle stability complex, in Drosophila. These studies demonstrate that myofibrils progressively unbundle in flies that lack Thin function through progressive costamere breakdown. Due to the high conservation of these structures in animals, a previously unknown role for TRIM32 proteins in myofibril stability is demonstrated (LaBeau-DiMenna, 2012).

TRIM32 deficiency impairs synaptic plasticity by excitatory-inhibitory imbalance via Notch pathway

Synaptic plasticity is the neural basis of physiological processes involved in learning and memory. Tripartite motif-containing 32 (TRIM32) has been found to play many important roles in the brain such as neural stem cell proliferation, neurogenesis, inhibition of nerve proliferation, and apoptosis. TRIM32 has been linked to several nervous system diseases including autism spectrum disorder, depression, anxiety, and Alzheimer's disease. However, the role of TRIM32 in regulating the mechanism of synaptic plasticity is still unknown. Electrophysiological studies using hippocampal slices revealed that long-term potentiation of CA1 synapses was impaired in TRIM32 deficient (KO) mice. Further research found that dendritic spines density, AMPA receptors, and synaptic plasticity-related proteins were also reduced. NMDA receptors were upregulated whereas GABA receptors were downregulated in TRIM32 deficient mice, explaining the imbalance in excitatory and inhibitory neurotransmission. This caused overexcitation leading to decreased neuronal numbers in the hippocampus and cortex. In summary, this study provides this maiden evidence on the synaptic plasticity changes of TRIM32 deficiency in the brain and proposes that TRIM32 relates the notch signaling pathway and its related mechanisms contribute to this deficit (Ntim, 2020).

TRIM32 regulates mitochondrial mediated ROS levels and sensitizes the oxidative stress induced cell death

Emerging evidence suggests that ubiquitin mediated post translational modification is a critical regulatory process involved in diverse cellular pathways including cell death. During ubiquitination, E3 ligases recognize target proteins and determine the topology of ubiquitin chains. Recruitment of E3 ligases to targets proteins under stress conditions including oxidative stress and their implication in cell death have not been systemically explored. This study characterized the role of TRIM32 as an E3 ligase in regulation of oxidative stress induced cell death. TRIM32 is ubiquitously expressed in cell lines of different origin and form cytoplasmic speckle like structures that transiently interact with mitochondria under oxidative stress conditions. The ectopic expression of TRIM32 sensitizes cell death induced by oxidative stress whereas TRIM32 knockdown shows a protective effect. The turnover of TRIM32 is enhanced during oxidative stress and its expression induces ROS generation, loss of mitochondrial transmembrane potential and decrease in complex-I activity. The pro-apoptotic effect was rescued by pan-caspase inhibitor or antioxidant treatment. E3 ligase activity of TRIM32 is essential for oxidative stress induced apoptotic cell death. Furthermore, TRIM32 decreases X-linked inhibitor of apoptosis (XIAP) level and overexpression of XIAP rescued cells from TRIM32 mediated oxidative stress and cell death. Overall, the results of this study provide the first evidence supporting the role of TRIM32 in regulating oxidative stress induced cell death, which has implications in numerous pathological conditions including cancer and neurodegeneration (Prajapati, 2020).

Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

Sonic hedgehog (SHH) signaling is crucial for the maintenance of the physiological self-renewal of granule neuron progenitor cells (GNPs) during cerebellar development, and its dysregulation leads to oncogenesis. However, how SHH signaling is controlled during cerebellar development is poorly understood. This study shows that Trim32, a cell fate determinant, is distributed asymmetrically in the cytoplasm of mitotic GNPs, and that genetic knockout of Trim32 keeps GNPs at a proliferating and undifferentiated state. In addition, Trim32 knockout enhances the incidence of medulloblastoma (MB) formation in the Ptch1 mutant mice. Mechanistically, Trim32 binds to Gli1, an effector of SHH signaling, via its NHL domain and degrades the latter through its RING domain to antagonize the SHH pathway. These findings provide a novel mechanism that Trim32 may be a vital cell fate regulator by antagonizing the SHH signaling to promote GNPs differentiation and a tumor suppressor in MB formation (Wang, 2020).

EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer

EGFR inhibition is an effective treatment in the minority of non-small cell lung cancer (NSCLC) cases harboring EGFR-activating mutations, but not in EGFR wild type (EGFRwt) tumors. This study demonstrates that EGFR inhibition triggers an antiviral defense pathway in NSCLC. Inhibiting mutant EGFR triggers Type I IFN-I upregulation via a RIG-I-TBK1-IRF3 pathway. The ubiquitin ligase TRIM32 associates with TBK1 upon EGFR inhibition, and is required for K63-linked ubiquitination and TBK1 activation. Inhibiting EGFRwt upregulates interferons via an NF-kappaB-dependent pathway. Inhibition of IFN signaling enhances EGFR-TKI sensitivity in EGFR mutant NSCLC and renders EGFRwt/KRAS mutant NSCLC sensitive to EGFR inhibition in xenograft and immunocompetent mouse models. Furthermore, NSCLC tumors with decreased IFN-I expression are more responsive to EGFR TKI treatment. It is proposed that IFN-I signaling is a major determinant of EGFR-TKI sensitivity in NSCLC and that a combination of EGFR TKI plus IFN-neutralizing antibody could be useful in most NSCLC patients (Gong, 2020).

TRIM32, but not its muscular dystrophy-associated mutant, positively regulates and is targeted to autophagic degradation by p62/SQSTM1

The tripartite motif (TRIM) proteins constitute a family of ubiquitin E3 ligases involved in a multitude of cellular processes, including protein homeostasis and autophagy. TRIM32 is characterized by six protein-protein interaction domains termed NHL, various point mutations in which are associated with limb-girdle-muscular dystrophy 2H (LGMD2H). This study shows that TRIM32 is an autophagy substrate. Lysosomal degradation of TRIM32 was dependent on ATG7 and blocked by knockout of the five autophagy receptors p62 (also known as SQSTM1), NBR1, NDP52 (also known as CALCOCO2), TAX1BP1 and OPTN, pointing towards degradation by selective autophagy. p62 directed TRIM32 to lysosomal degradation, while TRIM32 mono-ubiquitylated p62 on lysine residues involved in regulation of p62 activity. Loss of TRIM32 impaired p62 sequestration, while reintroduction of TRIM32 facilitated p62 dot formation and its autophagic degradation. A TRIM32(LGMD2H) disease mutant was unable to undergo autophagic degradation and to mono-ubiquitylate p62, and its reintroduction into the TRIM32-knockout cells did not affect p62 dot formation. In light of the important roles of autophagy and p62 in muscle cell proteostasis, these results point towards impaired TRIM32-mediated regulation of p62 activity as a pathological mechanisms in LGMD2H (Overa, 2019).

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFalpha)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. This study identified a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFalpha-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. these novel results collectively suggest that TRIM32 sensitizes TNFalpha-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFalpha signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism (Ryu, 20114).

Tripartite motif protein 32 facilitates cell growth and migration via degradation of Abl-interactor 2

Tripartite motif protein 32 (TRIM32) mRNA has been reported to be highly expressed in human head and neck squamous cell carcinoma, but the involvement of TRIM32 in carcinogenesis has not been fully elucidated. This study found by using yeast two-hybrid screening that TRIM32 binds to Abl-interactor 2 (Abi2), which is known as a tumor suppressor and a cell migration inhibitor, and TRIM32 was shown to mediate the ubiquitination of Abi2. Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility, whereas a dominant-negative mutant of TRIM32 lacking the RING domain inhibited the degradation of Abi2. In addition, it was found that TRIM32 suppresses apoptosis induced by cis-diamminedichloroplatinum (II) in HEp2 cell lines. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis, and resistance to anticancer drugs (Kano, 2008).

The interaction of Piasy with Trim32, an E3-ubiquitin ligase mutated in limb-girdle muscular dystrophy type 2H, promotes Piasy degradation and regulates UVB-induced keratinocyte apoptosis through NFkappaB

Protein inhibitors of activated STATs (PIAS) family members are ubiquitin-protein isopeptide ligase-small ubiquitin-like modifier ligases for diverse transcription factors. However, the regulation of PIAS protein activity in cells is poorly understood. Previously, it was reported that expression of Trim32, a RING domain ubiquitin-protein isopeptide ligase-ubiquitin ligase mutated in human limb-girdle muscular dystrophy type 2H (LGMD2H) and Bardet-Biedl syndrome, is elevated during mouse skin carcinogenesis, protecting keratinocytes from apoptosis induced by UVB and tumor necrosis factor-alpha (TNFalpha). This study reports that Trim32 interacts with Piasy and promotes Piasy ubiquitination and degradation. Ubiquitination of Piasy by Trim32 could be reproduced in vitro using purified components. Their interaction was induced by treatment with UVB/TNFalpha and involved redistribution of Piasy from the nucleus to the cytoplasm, where it accumulated in cytoplasmic granules that colocalized with Trim32. Piasy destabilization and ubiquitination required an intact RING domain in Trim32. The LGMD2H-associated missense point mutation prevented Trim32 binding to Piasy, and human Piasy failed to colocalize with human Trim32 in fibroblasts isolated from an LGMD2H patient. Trim32 expression increased the transcriptional activity of NFkappaB in epidermal keratinocytes, both under basal treatment and after UVB/TNFalpha treatment. Conversely, Piasy inhibited NFkappaB activity under the same conditions and sensitized keratinocytes to apoptosis induced by TNFalpha and UVB. These results indicate that, by controlling Piasy stability, Trim32 regulates UVB-induced keratinocyte apoptosis through induction of NFkappaB and suggests loss of function of Trim32 in LGMD2H (Albor, 2006).

Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11)

The identification of mutations in genes that cause human diseases has largely been accomplished through the use of positional cloning, which relies on linkage mapping. In studies of rare diseases, the resolution of linkage mapping is limited by the number of available meioses and informative marker density. One recent advance is the development of high-density SNP microarrays for genotyping. The SNP arrays overcome low marker informativity by using a large number of markers to achieve greater coverage at finer resolution. This study used SNP microarray genotyping for homozygosity mapping in a small consanguineous Israeli Bedouin family with autosomal recessive Bardet-Biedl syndrome (BBS; obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment) in which previous linkage studies using short tandem repeat polymorphisms failed to identify a disease locus. SNP genotyping revealed a homozygous candidate region. Mutation analysis in the region of homozygosity identified a conserved homozygous missense mutation in the TRIM32 gene, a gene coding for an E3 ubiquitin ligase. Functional analysis of this gene in zebrafish and expression correlation analyses among other BBS genes in an expression quantitative trait loci data set demonstrate that TRIM32 is a BBS gene. This study shows the value of high-density SNP genotyping for homozygosity mapping and the use of expression correlation data for evaluation of candidate genes and identifies the proteasome degradation pathway as a pathway involved in BBS (Chiang, 2006).

Search PubMed for articles about Drosophila Trim32

Albor, A., El-Hizawi, S., Horn, E. J., Laederich, M., Frosk, P., Wrogemann, K. and Kulesz-Martin, M. (2006). The interaction of Piasy with Trim32, an E3-ubiquitin ligase mutated in limb-girdle muscular dystrophy type 2H, promotes Piasy degradation and regulates UVB-induced keratinocyte apoptosis through NFkappaB. J Biol Chem 281(35): 25850-25866. PubMed ID: 16816390

Bawa, S., Brooks, D. S., Neville, K. E., Tipping, M., Sagar, M. A., Kollhoff, J. A., Chawla, G., Geisbrecht, B. V., Tennessen, J. M., Eliceiri, K. W. and Geisbrecht, E. R. (2020a). Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth. Elife 9. PubMed ID: 32223900

Bawa, S., Gameros, S., Baumann, K., Brooks, D. S., Kollhoff, J. A., Zolkiewski, M., David Re Cecconi, A., Panini, N., Russo, M., Piccirillo, R., Johnson, D. K., Kashipathy, M. M., Battaile, K. P., Lovell, S., Bouyain, S. E. A., Kawakami, J. and Geisbrecht, E. R. (2020b). Costameric integrin and Sarcoglycan protein levels are altered in a Drosophila model for Limb Girdle Muscular Dystrophy type 2H. Mol Biol Cell: mbcE20070453. PubMed ID: 33296226

Chiang, A. P., Beck, J. S., Yen, H. J., Tayeh, M. K., Scheetz, T. E., Swiderski, R. E., Nishimura, D. Y., Braun, T. A., Kim, K. Y., Huang, J., Elbedour, K., Carmi, R., Slusarski, D. C., Casavant, T. L., Stone, E. M. and Sheffield, V. C. (2006). Homozygosity mapping with SNP arrays identifies TRIM32, an E3 ubiquitin ligase, as a Bardet-Biedl syndrome gene (BBS11). Proc Natl Acad Sci U S A 103(16): 6287-6292. PubMed ID: 16606853

Gong, K., Guo, G., Panchani, N., Bender, M. E., Gerber, D. E., Minna, J. D., Fattah, F., Gao, B., Peyton, M., Kernstine, K., Mukherjee, B., Burma, S., Chiang, C. M., Zhang, S., Sathe, A. A., Xing, C., Dao, K. H., Zhao, D., Akbay, E. A. and Habib, A. A. (2020). EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer. Nat Cancer 1(4): 394-409. PubMed ID: 33269343

Kano, S., Miyajima, N., Fukuda, S. and Hatakeyama, S. (2008). Tripartite motif protein 32 facilitates cell growth and migration via degradation of Abl-interactor 2. Cancer Res 68(14): 5572-5580. PubMed ID: 18632609

LaBeau-DiMenna, E. M., Clark, K. A., Bauman, K. D., Parker, D. S., Cripps, R. M. and Geisbrecht, E. R. (2012). Thin, a Trim32 ortholog, is essential for myofibril stability and is required for the integrity of the costamere in Drosophila. Proc Natl Acad Sci U S A 109(44): 17983-17988. PubMed ID: 23071324

Lazzari, E. and Meroni, G. (2016). TRIM32 ubiquitin E3 ligase, one enzyme for several pathologies: From muscular dystrophy to tumours. Int J Biochem Cell Biol 79: 469-477. PubMed ID: 27458054

Li, H., Rai, M., Buddika, K., Sterrett, M. C., Luhur, A., Mahmoudzadeh, N. H., Julick, C. R., Pletcher, R. C., Chawla, G., Gosney, C. J., Burton, A. K., Karty, J. A., Montooth, K. L., Sokol, N. S. and Tennessen, J. M. (2019). Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase cooperatively regulate growth and carbohydrate metabolism during Drosophila melanogaster larval development. Development 146(17). PubMed ID: 31399469

Liu, J., Zhang, C., Wang, X. L., Ly, P., Belyi, V., Xu-Monette, Z. Y., Young, K. H., Hu, W. and Feng, Z. (2014). E3 ubiquitin ligase TRIM32 negatively regulates tumor suppressor p53 to promote tumorigenesis. Cell Death Differ 21(11): 1792-1804. PubMed ID: 25146927

Ntim, M., Li, Q. F., Zhang, Y., Liu, X. D., Li, N., Sun, H. L., Zhang, X., Khan, B., Wang, B., Wu, Q., Wu, X. F., Walana, W., Khan, K., Ma, Q. H., Zhao, J. and Li, S. (2020). TRIM32 deficiency impairs synaptic plasticity by excitatory-inhibitory imbalance via Notch pathway. Cereb Cortex 30(8): 4617-4632. PubMed ID: 32219328

Overa, K. S., Garcia-Garcia, J., Bhujabal, Z., Jain, A., Overvatn, A., Larsen, K. B., Deretic, V., Johansen, T., Lamark, T. and Sjottem, E. (2019). TRIM32, but not its muscular dystrophy-associated mutant, positively regulates and is targeted to autophagic degradation by p62/SQSTM1. J Cell Sci 132(23). PubMed ID: 31685529

Prajapati, P., Gohel, D., Shinde, A., Roy, M., Singh, K. and Singh, R. (2020). TRIM32 regulates mitochondrial mediated ROS levels and sensitizes the oxidative stress induced cell death. Cell Signal 76: 109777. PubMed ID: 32918979

Ryu, Y. S., Lee, Y., Lee, K. W., Hwang, C. Y., Maeng, J. S., Kim, J. H., Seo, Y. S., You, K. H., Song, B. and Kwon, K. S. (2011). TRIM32 protein sensitizes cells to tumor necrosis factor (TNFalpha)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP). J Biol Chem 286(29): 25729-25738. PubMed ID: 21628460

Tennessen, J. M., Baker, K. D., Lam, G., Evans, J. and Thummel, C. S. (2011). The Drosophila estrogen-related receptor directs a metabolic switch that supports developmental growth. Cell Metab 13(2): 139-148. PubMed ID: 21284981

Tennessen, J. M., Bertagnolli, N. M., Evans, J., Sieber, M. H., Cox, J. and Thummel, C. S. (2014). Coordinated metabolic transitions during Drosophila embryogenesis and the onset of aerobic glycolysis. G3 (Bethesda) 4(5): 839-850. PubMed ID: 24622332

Tixier, V., Bataille, L., Etard, C., Jagla, T., Weger, M., Daponte, J. P., Strahle, U., Dickmeis, T. and Jagla, K. (2013). Glycolysis supports embryonic muscle growth by promoting myoblast fusion. Proc Natl Acad Sci U S A 110(47): 18982-18987. PubMed ID: 24191061

Tocchini, C. and Ciosk, R. (2015). TRIM-NHL proteins in development and disease. Semin Cell Dev Biol 47-48: 52-59. PubMed ID: 26514622

Vishal, K., Bawa, S., Brooks, D., Bauman, K. and Geisbrecht, E. R. (2018). Thin is required for cell death in the Drosophila abdominal muscles by targeting DIAP1. Cell Death Dis 9(7): 740. PubMed ID: 29970915

Wang, M., Luo, W., Zhang, Y., Yang, R., Li, X., Guo, Y., Zhang, C., Yang, R. and Gao, W. Q. (2020). Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling. Cell Death Differ 27(4): 1286-1299. PubMed ID: 31527798

Watanabe, M. and Hatakeyama, S. (2017). TRIM proteins and diseases. J Biochem 161(2): 135-144. PubMed ID: 28069866

Zhu, J. and Thompson, C. B. (2019). Metabolic regulation of cell growth and proliferation. Nat Rev Mol Cell Biol 20(7): 436-450. PubMed ID: 30976106

Zirin, J., Cheng, D., Dhanyasi, N., Cho, J., Dura, J. M., Vijayraghavan, K. and Perrimon, N. (2013). Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles. Dev Biol 383(2): 275-284. PubMed ID: 24051228

date revised: 30 January 2021

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