Author response: Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cell 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. Herein, we identify 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, our results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth. Introduction The metabolism of all cells must adapt to meet the energetic and biosynthetic needs of growth and homeostasis (Lloyd, 2013; Zhu and Thompson, 2019). 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 (Lloyd, 2013). 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 (Zhu and Thompson, 2019). Striking this delicate balance between degradative and biosynthetic processes requires the integration of extracellular and intracellular information by complex signaling networks. The mechanisms by which cell proliferation and tissue growth rewire metabolism to enhance biosynthesis are diverse and complex (Lunt and Vander Heiden, 2011). 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 and Thompson, 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 et al., 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 et al., 2014b). 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. 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 (Tocchini and Ciosk, 2015; Watanabe and Hatakeyama, 2017). 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 and Meroni, 2016; Tocchini and Ciosk, 2015; Watanabe and Hatakeyama, 2017). A single mutation in the B-box region of TRIM32 causes the multisystemic disorder Bardet-Biedl syndrome (BBS) (Chiang et al., 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) (Borg et al., 2009; Frosk et al., 2005; Lazzari et al., 2019; Nectoux et al., 2015; Neri et al., 2013; Schoser et al., 2005; Servián-Morilla et al., 2019). 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 (Albor et al., 2006; Cohen et al., 2014; Cohen et al., 2012; Kudryashova et al., 2005; Locke et al., 2009; Volodin et al., 2017) or the prevention of satellite cell senescence (Kudryashova et al., 2012; Mokhonova et al., 2015; Servián-Morilla et al., 2019), consistent with a role for TRIM32 in LGMD2H. However, additional polyubiquitinated substrates, including p53, Abi2, Piasy, XIAP, and MYCN, are implicated in tumorigenesis (Albor et al., 2006; Izumi and Kaneko, 2014; Kano et al., 2008; Liu et al., 2014; Ryu et al., 2011). Importantly, TRIM32 protein levels are upregulated in multiple tumor types, suggesting that TRIM32 is a key player in growth regulation (Horn et al., 2004; Ito et al., 2017; Zhao et al., 2018). 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 (Arama et al., 2000; Edwards et al., 2003). Here, we provide a novel mechanism for TRIM32 in cell growth. Our 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. Results TRIM32 binds to glycolytic enzymes While NHL domain-containing proteins can interact with both RNAs and proteins (Tocchini and Ciosk, 2015; Watanabe and Hatakeyama, 2017), few bona fide TRIM32 binding partners have been identified. Causative mutations in human LGMD2H cluster in the NHL repeats (Figure 1A; Borg et al., 2009; Cossée et al., 2009; Frosk et al., 2005; Lazzari et al., 2019; Nectoux et al., 2015; Neri et al., 2013; Schoser et al., 2005), suggesting that this region may mediate protein-protein interactions important in disease prevention. We previously showed that mutations in Drosophila TRIM32 also show progressive larval muscle degeneration, despite modest sequence identity (~42%) across the NHL domains (LaBeau-DiMenna et al., 2012). To gain molecular-level insight into the functional conservation between the NHL regions in fly and mammalian TRIM32, we obtained a crystal structure of TRIM32_NHL at 2.6 Å resolution (Rwork/Rfree = 18.2/22.8%) (Figure 1—figure supplement 1A–C; Figure 1—source data 1). The structure was solved by molecular replacement, using the NHL-repeat region of the Drosophila Brat protein as a search model (PDB Code 1Q7F) (Edwards et al., 2003). This structure features a six-bladed β-propeller, where each NHL repeat is comprised of four antiparallel β-strands (Figure 1B; Figure 1—figure supplement 1C). We then used the TRIM32_NHL structure to construct a model for mouse TRIM32_NHL using SWISS-MODEL. Indeed, the superimposition of these two structures demonstrate that the NHL region of Drosophila TRIM32 is a faithful model to understand NHL function (Figure 1B). Figure 1 with 1 supplement see all Download asset Open asset The NHL region of Drosophila TRIM32 is structurally conserved. (A) Schematic showing the RING, B-box, coiled-coil, and NHL domains in TRIM32. (B) Superimposed protein structures of the six NHL repeats in Drosophila (magenta) and Mus musculus (blue) TRIM32. Each NHL repeat consists of four antiparallel beta sheets that are arranged toroidally around a central axis. Mouse NHL has two additional loops (asterisks) not present in the fly protein. The positions and orientation of both R394/R1114 and D487/D1211 are identical between Mus musculus and Drosophila (orange). (C) The glycolytic pathway. Peptides corresponding to enzymes that co-purified with TRIM32_NHL are shown in blue. Figure 1—source data 1 X-ray diffraction data collection and refinement statistics. https://cdn.elifesciences.org/articles/52358/elife-52358-fig1-data1-v1.docx Download elife-52358-fig1-data1-v1.docx Figure 1—source data 2 Proteins identified via MS that co-purify with TRIM32-NHL Experiment #1 (1418) Experiment #2 (1426.1) Experiment #3 (1426.2). https://cdn.elifesciences.org/articles/52358/elife-52358-fig1-data2-v1.docx Download elife-52358-fig1-data2-v1.docx To identify TRIM32_NHL-interacting proteins, we performed in vivo pulldowns followed by mass spectrometry (MS) to uncover associated peptides. Briefly, the C-terminal region of Drosophila TRIM32 containing all six NHL domains (AA 1061–1353) was fused in-frame with an N-terminal Glutathione S-transferase (GST) tag. This GST_TRIM32_NHL protein was expressed, purified, and incubated with third instar larval (L3) lysates. These resulting protein complexes were subjected to MS to detect peptide fragments that co-purify with TRIM32_NHL. In addition to the recovery of our bait protein, we detected peptides corresponding to Tropomyosin (Tm) and Troponin T (TnT) (Figure 1—source data 2), which are both known polyubiquitinated substrates of mammalian TRIM32 (Cohen et al., 2012). Surprisingly, glycolytic enzymes were also enriched in the list of possible NHL-binding proteins (Figure 1C; Figure 1—source data 2) and were further evaluated for physical interactions with TRIM32. We utilized two independent methods to validate candidate protein interactions with TRIM32. First, an in vitro binding assay using purified proteins was developed with Tropomyosin 2 (Tm2) as a positive control. Indeed, incubation of TRIM32_NHL together with His-tagged Tm2 confirmed a physical interaction between these two proteins (Figure 2—figure supplement 1A). This assay was then used to test for physical interactions with candidate glycolytic enzymes. To this end, we expressed and purified His-tagged Aldolase (Ald) or His-tagged Phosphoglycerate mutase 78 (Pglym), both of which were low or absent in control pulldowns. Both Ald and Pglym were found to directly interact with the NHL domains of TRIM32 (Figure 2A,B). Note that no binding was detected between TRIM32_NHL and the His-tagged control protein SCIN, ruling out non-specific binding of the His tag. To extend these findings, we utilized immunoprecipitation experiments to test for in vivo interactions with full-length TRIM32. As expected, immunoprecipitation of TRIM32 successfully pulled-down Tm as an interacting protein at the predicted molecular weight of ~37 kD (Figure 2—figure supplement 1B). We also confirmed that both Ald and Pglym co-immunoprecipitate with anti-TRIM32 antibody. Interestingly, the bands corresponding to these proteins migrated at a higher molecular weight than the expected size present in input lysates (Figure 2C,D; asterisk), suggestive of a post-translational modification. Two approaches were taken to confirm the specificity of the slower migrating bands that correspond to Ald or Pglym. First, we concentrated L3 lysates before SDS-PAGE analysis. After overexposure of the western blots, faint bands corresponding to the higher molecular weight forms of Ald or Pglym were present (Figure 2C,D; asterisk). We also immunoprecipitated TRIM32 from larval lysates and probed for Ald or Pglym using antibodies raised against the orthologous human proteins (ALD or PGAM1). While these antibodies did not cross react well in input lysates, there was an obvious band corresponding to the higher molecular weight forms of both Ald and Pglym after pulling down TRIM32 (Figure 2—figure supplement 1D,E). Collectively, these data demonstrate that Ald and Pglym interact with the NHL domain of TRIM32 and in vivo, a subpopulation of these post-translationally modified glycolytic enzymes can be found in a complex with TRIM32. Figure 2 with 1 supplement see all Download asset Open asset Drosophila TRIM32 physically interacts with the glycolytic enzymes Ald and Pglym78. (A,B) In vitro binding assays. Untagged TRIM32_NHL was incubated with either the His-tagged SCIN control protein or the His-tagged candidate proteins Ald (A) or Pglym (B). After washing in 300 mM NaCl and 0.1% Triton, each of these protein complexes was separated by SDS-PAGE followed by Coomassie staining. Ald and Pglym proteins directly bind the NHL region of TRIM32, while no interaction with the His-SCIN control protein is observed. (C,D) Western blotting with antibodies against Drosophila Ald (dAld; C) or Drosophila Pglym (dPglym; D) detects higher molecular weight bands (asterisk) upon immunoprecipitation of Drosophila TRIM32, but not in control lanes. The observed molecular weights of Ald or Pglym in input larval lysates (~30 µg) is predominant over a higher migrating form that can be visualized after overexposure of blots with concentrated lysate (~300 µg). (E,F) Western blots showing that mutations in tn reduce Ald (E) or Pglym (F) protein levels ~ 50% quantitated relative to ATP5α in L3 larvae. N = 3. Mean +/- SD (*, p<0.05). The majority of TRIM32 studies have focused on the biological impact of polyUb chain addition onto substrates (Cohen et al., 2012; Kudryashova et al., 2005; Liu et al., 2014; Locke et al., 2009; Mokhonova et al., 2015). This modification targets a protein for proteasomal degradation and usually increases protein substrate levels in the absence of its E3 counterpart. Indeed, the known mammalian substrate Tm binds to TRIM32_NHL (Cohen et al., 2012) and accordingly, Drosophila Tm was modestly increased in larvae mutant for tn (Figure 2—figure supplement 1C). Strikingly, this same loss of TRIM32 resulted in a ~ 50% decrease in the protein levels of Ald or Pglym78 (Figure 2E,F). This downregulation was not due to a decrease in TRIM32-mediated transcription as mRNA levels of Ald or Pglym were not altered in tn-/- (Figure 2—figure supplement 1F,G). Taken together, these data highlight a unique role for TRIM32 in the stabilization of glycolytic enzyme levels. Loss of TRIM32 disrupts glycolytic metabolism in larval muscles To understand the biological implications for the physical interaction between TRIM32 and the glycolytic enzymes Ald and Pglym, we profiled 85 metabolites present in WT or tn-/- L3 larvae. Principal component analysis (PCA) revealed a distinct separation between WT and tn-/- experimental groups (Figure 3—figure supplement 1A). Examination of individual metabolites upon loss of TRIM32 showed a decrease in the terminal glycolytic products pyruvate and lactate as well as significant depletion of the glucose-derived metabolites glycerol-3-phosphate and 2-hydroxyglutarate (Figure 3A,B). These metabolic changes suggest that TRIM32 is required for sustaining the conversion of glucose to pyruvate and lactate in L3 larvae. Figure 3 with 1 supplement see all Download asset Open asset Loss of TRIM32 decreases glycolytic flux and reduces muscle tissue size. (A) Volcano plot illustrating fold change (FC) (log base 2) compared with p-value (- log base 10) between WT and tn-/- L3 larvae. Vertical line represents FC >1.5. Horizontal line depicts a significance level p<0.05. Metabolites that are reduced in tn-/- larvae include indicators of glycolytic flux (green) and amino acids (blue). Metabolites in gray are significant, but exhibit a FC <1.5. (B) Box and whisker plot of terminal glycolytic metabolites significantly reduced upon loss of TRIM32. N = 6. (C–F) Ventral longitudinal muscles 3 (VL3) and 4 (VL4) stained with phalloidin to visualize F-actin (green). (C,D) The stereotypical morphology of WT muscles is not altered as overall muscle size increases from the L2 (C) to the L3 (D) stage. (E,F) In addition to sarcomeric disorganization, the VL3 and VL4 muscles are noticeably smaller in tn-/- larvae during L2 (E) and L3 (F) development. Muscle attachment sites (MASs) are denoted by yellow lines. (G) Scatter plot depicting VL3 muscle diameter. The diameter of WT muscles increase from the L2 to the L3 stage. This cell size increase is abolished in tn-/-. N ≥ 8. (H) Bar graph shows that ECAR measurements are decreased in isolated tn-/- muscle carcasses compared to WT. N ≥ 4. (I) Analysis of the glycolytic rate in WT or tn-/- muscle tissue after subtraction of mitochondrial-produced acidification. This PER is diminished upon loss of TRIM32. N = 4. (J) NADH lifetime image comparison of WT and tn-/- muscles. Box and whisker plot shows WT muscles have significantly lower NADH lifetime, indicative of higher glycolytic flux, than tn-/-. N = 5. Mean +/- SD. (****, p<0.001; ***, p<0.01; *, p<0.05; n.s., not significant). Scale bars: 25 µm (C,E), 50 µm (D,F). The production of pyruvate at the end of glycolysis has two fates, either a reduction to lactate or oxidation to CO2 in the mitochondrion (Mookerjee et al., 2015; TeSlaa and Teitell, 2014). To determine if reduced lactate levels in tn-/- results in increased respiration and therefore elevated ATP synthesis, we assessed these two outputs of overall energy metabolism in control or mutant whole larvae. Respirometry analysis revealed a modest increase in CO2 production upon loss of TRIM32 (Figure 3—figure supplement 1B). However, ATP levels were significantly decreased in tn-/- larvae (Figure 3—figure supplement 1C). Given that different larval tissues may have altered metabolic profiles that influence outputs of whole body metabolism, we sought to assess glycolytic activity in individual tissues. A unique aspect of Drosophila larval development is the 200-fold increase in body size from first instar (L1) larvae through the L3 stage. In order to support high levels of biomass accumulation, glycolytic metabolism is increased prior to larval growth (Tennessen et al., 2011; Tennessen et al., 2014b). One larval tissue that requires high glycolytic activity is the somatic musculature, where WT body wall muscles undergo dramatic growth as development proceeds from second instar larvae (L2) (Figure 3C,G) to the L3 stage (Figure 3D,G). We, and others, previously reported a loss of structural integrity in TRIM32-deficient muscles et al., 2013; et al., 2012) and that the associated reduction in muscle size was a of progressive tissue While multiple independent of tn have been shown to smaller muscles et al., 2013; et al., of tn-/- confirmed a decrease in larval muscle diameter in both L2 (Figure and L3 (Figure loss of TRIM32 the growth of muscles during larval development. As an of glycolytic we the extracellular rate in WT or tn-/- muscle carcasses using the a to extracellular is lactate into during glycolysis (Mookerjee and 2015; TeSlaa and Teitell, we and observed a reduction in ECAR upon loss of TRIM32 (Figure While such as a of muscles at a single For measurements of glycolytic flux, the was used to the rate This assay specifically provides a of from glycolysis by for both the production of from and for the of the assay Briefly, the assay ECAR of the without addition of the chain and and after addition of the glycolytic The end result of the assay is a of rate due to glycolytic Our analysis revealed that glycolytic metabolism in tn-/- muscle carcasses as compared with WT (Figure our that TRIM32 is required to support glycolytic To validate that loss of TRIM32 results in decreased glycolytic flux, we directly NADH levels muscle tissues using lifetime which the of this to visualize NADH levels in cellular et al., et al., 2009; et al., 2011; et al., 2017). the lifetime of NADH required for NADH to to is NADH is to enzymes than in the this assay can intracellular NADH et al., 2005; et al., This analysis revealed that WT muscles by showed a lifetime than tn-/- muscles (Figure NADH is in glycolytic cells, we confirm a reduction in the glycolytic profile upon loss of TRIM32. These taken together, support the that TRIM32 glycolytic flux in larval muscle tissue. One of tn mutant is the that the observed muscle result from loss of TRIM32 in other tissues. To out TRIM32-mediated we utilized approaches and First, we that of independent tn mRNA with the muscle smaller muscles (Figure et al., 2016; et al., 2013; et al., 2012). of TRIM32 in a single muscle each was to reduce muscle cell size (Figure As a to glycolytic we levels in muscle Consistent with our the relative of lactate levels was decreased in tn-/- muscles (Figure of tn in muscle tissue decreased lactate while this same reduction did not upon TRIM32 in (Figure Here, we that TRIM32-mediated regulation of glycolysis in muscle tissue is cell Figure 4 Download asset Open asset Muscle are cell and can be upon stabilization of glycolytic enzyme levels. of TRIM32 in muscle tissue decreases muscle size and reduces lactate levels. muscles in a of the (A) control muscles WT. of tn in all muscles with (B) or muscle using the (C) show a reduction in muscle size (D) of tn mRNA with independent in muscle tissue control of the show reduced VL3 muscle diameter compared to VL3 muscles. N ≥ (E) Bar graph a cell role for TRIM32 in muscle tissue. levels in muscle carcasses are decreased upon loss of TRIM32 in all tissues. of tn in muscle, but not neuronal reduces the of N 8. expression of TRIM32 or in a tn-/- the loss of muscle muscle and glycolytic protein levels. (F) Scatter plot shows that the reduced VL3 muscle diameter upon loss of TRIM32 is upon expression of TRIM32 or in muscle tissue. N ≥ The to body wall muscles in tn-/- causes expression of TRIM32 or muscle (G) of the (H) of by a and whisker N = Western blots showing the relative of Ald or Pglym protein relative to the ATP5α control. Both Ald and Pglym protein levels are upon TRIM32 or expression in muscle tissue compared to tn-/-. N = 3. Mean +/- SD (****, p<0.001; ***, p<0.01; p<0.05; *, p<0.01; n.s., not significant). is a that as a switch in to genes required for glycolysis during larval growth (Tennessen et al., 2011). we that upregulation of metabolism genes via may muscle growth and As a positive we expressed a for TRIM32 in tn-/- muscle tissue and found that the muscle diameter was to WT (Figure This result also confirmed the cell of TRIM32. of in tn-/- muscles not muscle size (Figure but also the functional associated with the of tn-/- to body wall muscles during (Figure et al., 2013; et al., 2012). Importantly, protein levels of Ald and Pglym were upon expression of in tn-/- muscles (Figure that of glycolytic protein levels is to TRIM32-mediated growth TRIM32 amino acid Our analysis revealed that loss of TRIM32 not disrupts the production of glycolytic but also induces a significant decrease in of the amino acids (Figure These changes in amino acid are due to both decreased synthesis and increased (Figure 3—figure supplement as we observed that tn-/- decrease in not and levels which are from but also a reduction in the amino acids and Moreover, loss of TRIM32 also a significant depletion of which is both from and into Overall, the profile of tn-/- that of glucose results in depletion of larval amino acid the that decreased amino acid availability to the tn-/- muscle We this by the of both mutant and control larvae with amino acid As an we first if the tn-/- larval enhanced to Indeed, on the muscle diameter of tn mutants was smaller than muscles of control larvae raised on the same (Figure In contrast, mutant larvae a of yeast or with all amino acids no decrease in muscle diameter (Figure We observed results in the context of L3 body mass and muscle during the larval to whereby tn-/- raised on an compared with WT control larvae (Figure of the larval with yeast or amino acids, however, both in tn-/- (Figure These results suggest that tn-/- mutants are to dietary amino acids, consistent with the smaller amino acid size observed upon loss of TRIM32. Figure Download asset Open asset acid supplementation is to tn-/- muscle mass. (A) Box and whisker showing the relative of individual amino acids in L3 larvae with a FC or FC that are significantly reduced upon loss of TRIM32. N = 6. of WT (B) or tn-/- L3 muscles stained for depicts two complete and lower on the VL3 and VL4 muscles. are denoted by yellow lines. (B) of WT on as a (C) in larvae for TRIM32 are raised on of the reduced muscle diameter is observed in tn-/- muscles with yeast (D) or amino acids (E) compared to tn-/- muscles (F) Scatter plot showing the diameter of muscle VL3 in WT or tn-/- to the N ≥ (G) body mass measurements of WT or tn-/- L3 larvae. were