New Members of the Mammalian Glycerophosphodiester Phosphodiesterase Family: GDE4 AND GDE7 PRODUCE LYSOPHOSPHATIDIC ACID BY LYSOPHOSPHOLIPASE D ACTIVITY*

The known mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) hydrolyze glycerophosphodiesters. In this study, two novel members of the mammalian GP-PDE family, GDE4 and GDE7, were isolated, and the molecular basis of mammalian GP-PDEs was further explored. The GDE4 and GDE7 sequences are highly homologous and evolutionarily close. GDE4 is expressed in intestinal epithelial cells, spermatids, and macrophages, whereas GDE7 is particularly expressed in gastro-esophageal epithelial cells. Unlike other mammalian GP-PDEs, GDE4 and GDE7 cannot hydrolyze either glycerophosphoinositol or glycerophosphocholine. Unexpectedly, both GDE4 and GDE7 show a lysophospholipase D activity toward lysophosphatidylcholine (lyso-PC). We purified the recombinant GDE4 and GDE7 proteins and show that these enzymes can hydrolyze lyso-PC to produce lysophosphatidic acid (LPA). Further characterization of purified recombinant GDE4 showed that it can also convert lyso-platelet-activating factor (1-O-alkyl-sn-glycero-3-phosphocholine; lyso-PAF) to alkyl-LPA. These data contribute to our current understanding of mammalian GP-PDEs and of their physiological roles via the control of lyso-PC and lyso-PAF metabolism in gastrointestinal epithelial cells and macrophages.     

A Novel Glycerophosphodiester Phosphodiesterase,GDE5, Controls Skeletal Muscle Development via a Non-enzymatic Mechanism

Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5ΔC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5ΔC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.     

Genomic organization, characterization, and molecular 3D model of GDE1, a novel mammalian glycerophosphoinositol phosphodiesterase

Glycerophosphodiester phosphodiesterase (GDPD) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. A mammalian glycerophosphoinositol phosphodiesterase, GDE1/MIR16, was recently identified as an interacting protein of the regulator of G protein signaling 16 (RGS16) providing a link between phosphoinositide metabolism and G protein signal transduction. To further understand the function and properties of human GDE1, we determined its genomic organization and its biochemical and structural characteristics. GDE1 encodes a 331-residue protein with two hydrophobic domains and contains a GDE domain that shares strong homologies with GDE1-related proteins as well as bacterial GDPDs. The human GDE1 gene is located on chromosome 16p12-p11.2 and contains six exons and five introns. A molecular 3D model, which was built based on the crystal structure of Escherichia coli GDPD (1YDY), provides the first structural information of human GDE1 and suggests a TIM barrel core as typically found in bacterial GDPDs. Furthermore, a model of the putative catalytic motif within the GDE domain was nearly identical to the corresponding domain of GDPD and highlights the individual core residues Glu97, Asp99, and His112, which are crucial to maintaining GDE1 catalytic activity. These studies provide important new insights into understanding the function of GDE1 and GDE1-related proteins.     

The Developmentally Regulated Osteoblast Phosphodiesterase GDE3 Is Glycerophosphoinositol-specific and Modulates Cell Growth

The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells.The glycerophosphodiester phosphodiesterases (GP-PDEs)⁵ were initially characterized in bacteria, where they have functional roles for production of metabolic carbon and phosphate sources from glycerophosphodiesters (1, 2) and in adherence to and degradation of mammalian host-cell membranes (3). The GP-PDEs have a catalytic region of 56 amino acids (4). After their characterization in bacteria, mammalian glycerophosphodiesterases were identified, with the definition of a family of seven members (5). The first of these, GDE1, is an interactor of regulator of G-protein signaling (RGS)16, and was subsequently defined as a GP-PDE regulated by G-protein signaling (4). Indeed, GDE1 expression in HEK293T cells showed increased enzymatic activity upon α/β-adrenergic and lysophospholipid receptor stimulation (4). The second member, GDE2, was isolated by homology searches in neuronal tissues and its physiological role involves neuronal differentiation (6, 7). In contrast, GDE3 has been characterized as a marker of osteoblast differentiation and was isolated through a differential display method (8). GDE4 was isolated only recently with three-dimensional modeling defining it as a GP-PDE, although no functional activity has been correlated to its expression (9). The remaining members were cloned following data base searches, with further studies required for the definition of their properties (5). The diversity among these family members, in terms of tissue distribution, subcellular localization, and substrate specificity, suggests they selectively regulate biological functions and have distinct physiological roles (5).The only GP-PDE activity that has been biochemically characterized to date followed GDE1 overexpression in HEK293T cells, which showed a selectivity for the glycerophosphoinositols (GPIs) as substrate (4), in contrast to the bacterial GP-PDEs that show broad substrate specificities with respect to the alcohol moiety of the glycerophosphodiesterases (1, 2). The GPIs are naturally occurring, biologically active metabolites of the phosphoinositides that were originally investigated in the context of Ras-transformed cells (10). They are present in virtually all cell types, where their intracellular levels can also be modulated according to cell activation, differentiation, and development (Refs. 11 and 12 and references therein). Recently, glycerophosphoinositol (GroPIns) was characterized as a mediator of purinergic and adrenergic regulation of PCCl₃ thyroid cell proliferation (13), while GroPIns 4-phosphate (GroPIns4P) has been shown to induce reorganization of the actin cytoskeleton in fibroblasts and in T-lymphocytes, by promoting a sustained and robust activation of the Rho GTPases (14–16).The GPIs appear to rapidly equilibrate across the plasma membrane when added exogenously to cells, to exert their actions within the cell (12). The plasma membrane transporter for GroPIns characterized in yeast is the protein GIT1 (17), with one of its orthologs in mammalian cells identified as the human permease Glut2 (18). This specific transporter has been proposed to mediate both GroPIns uptake and release, which depends on the GroPIns concentration gradient across the plasma membrane. Under physiological conditions, this gradient can arise from the formation of GPIs from the phosphoinositides inside cells following activation of a specific isoform of phospholipase A₂, PLA₂IVα (13, 19).The release of the GPIs into the extracellular medium can affect their paracrine targets (16) or initiate their catabolism. This is supported by our characterization of GDE1 activity, and now of GDE3 activity, both of which show a substrate selectivity toward GroPIns, and catalytic activity after heterologous expression that can only be monitored in the extracellular space. Interestingly, GDE3 activity appears to be related to modulation of osteoblast functions, delineating a role for GDE3 in promoting osteoblast differentiation, and mainly regulating osteoblast proliferation.     

Novel membrane protein containing glycerophosphodiester phosphodiesterase motif is transiently expressed during osteoblast differentiation

Osteoblast maturation is a multistep series of events characterized by an integrated cascade of gene expression that are accompanied by specific phenotypic alterations. To find new osteoblast-related genes we cloned differentially expressed cDNAs characteristic of specific differentiation stages in the mouse osteoblast-like MC3T3-E1 cells by a differential display method. We identified a novel cDNA encoding a putative glycerophosphodiester phosphodiesterase, GDE3, which specifically was expressed at the stage of matrix maturation. Interestingly, the deduced amino acid sequence contains 539 amino acids including seven putative transmembrane domains and a glycerophosphodiester phosphodiesterase region in one of the extracellular loops. Northern blot analysis revealed that GDE3 was also expressed in spleen as well as primary calvarial osteoblasts and femur. We next transfected HEK293T cells with GDE3 with green fluorescent protein fused to the C terminus. The green fluorescent protein-fused protein accumulated at the cell periphery, and the transfected cells overexpressing the protein changed from a spread form to rounded form with disappearance of actin filaments. Immunofluorescence staining with GDE3 antibody and phalloidin in MC3T3-E1 cells indicated that endogenous GDE3 might be co-localized with the actin cytoskeleton. To identify a role for GDE3 in osteoblast differentiation, MC3T3-E1 cells stably expressing the full-length protein were constructed. Expression of GDE3 showed morphological changes, resulting in dramatic increases in alkaline phosphatase activity and calcium deposit. These results suggest that GDE3 might be a novel seven-transmembrane protein with a GP-PDE-like extracellular motif expressed during the osteoblast differentiation that dramatically accelerates the program of osteoblast differentiation and is involved in the morphological change of cells.     

Isolation,characterization and molecular 3D model of human GDE4, a novel membrane protein containing glycerophosphodiester phosphodiesterase domain

As a transmembrane protein family, glycerophosphodiester phosphodiesterase (GDPD/GDE) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. To date, seven mammalian GDEs have been virtually cloned or predicted by bioinformatics analysis, however, GDE4 has not been molecular isolated and characterized in mammal. Here we report molecular cloning of human GDE4 encoding cDNA sequence, which is 945 base pairs long encoding a 314-amino acid protein with 2 transmembrane regions and a GDE motif. The human GDE1 gene is located on chromosome 19q22 and contains ten exons and nine introns. A molecular 3-D model provides the first structural information of human GDE4 and suggests a triose-phosphate-isomerase barrel core as typically found in bacterial GDPDs. Furthermore, a model of the putative catalytic residues highlights that the individual core residues Glu72, Asp74, and His87 are crucial to maintaining GDE4 catalytic activity. Western blotting shows that human GDE4 is a 36 kDa protein. Subcellular localization of GDE4 tagged with enhanced green fluorescence protein is in the cytoplasm, especially accumulated in the perinuclear region and the cell periphery. Moreover, over-expression of GDE4 did not induce neurite formation or change cell morphology. These results indicate GDE4 protein is a member of the GDE family and suggest it may play different roles from other members of GDE family.     

Isolation, characterization and molecular 3D model of human GDE4, a novel membrane protein containing glycerophosphodiester phosphodiesterase domain

As a transmembrane protein family, glycerophosphodiester phosphodiesterase (GDPD/GDE) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. To date, seven mammalian GDEs have been virtually cloned or predicted by bioinformatics analysis, however, GDE4 has not been molecular isolated and characterized in mammal. Here we report molecular cloning of human GDE4 encoding cDNA sequence, which is 945 base pairs long encoding a 314-amino acid protein with 2 transmembrane regions and a GDE motif. The human GDE1 gene is located on chromosome 19q22 and contains ten exons and nine introns. A molecular 3-D model provides the first structural information of human GDE4 and suggests a triose-phosphate-isomerase barrel core as typically found in bacterial GDPDs. Furthermore, a model of the putative catalytic residues highlights that the individual core residues Glu72, Asp74, and His87 are crucial to maintaining GDE4 catalytic activity. Western blotting shows that human GDE4 is a 36 kDa protein. Subcellular localization of GDE4 tagged with enhanced green fluorescence protein is in the cytoplasm, especially accumulated in the perinuclear region and the cell periphery. Moreover, over-expression of GDE4 did not induce neurite formation or change cell morphology. These results indicate GDE4 protein is a member of the GDE family and suggest it may play different roles from other members of GDE family.     

Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.     

MIR125B1 represses the degradation of the PML-RARA oncoprotein by an autophagy-lysosomal pathway in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)-associated PML-RARA fusion gene. We have previously found that MIR125B1 is highly expressed in patients with APL and may be associated with disease pathogenesis; however, the mechanism by which MIR125B1 exerts its oncogenic potential has not been fully elucidated. Here, we demonstrated that MIR125B1 abundance correlates with the PML-RARA status. MIR125B1 overexpression enhanced PML-RARA expression and inhibited the ATRA-induced degradation of the PML-RARA oncoprotein. RNA-seq analysis revealed a direct link between the PML-RARA degradation pathway and MIR125B1-arrested differentiation. We further demonstrated that the MIR125B1-mediated blockade of PML-RARA proteolysis was regulated via an autophagy-lysosomal pathway, contributing to the inhibition of APL differentiation. Furthermore, we identified DRAM2 (DNA-damage regulated autophagy modulator 2), a critical regulator of autophagy, as a novel target that was at least partly responsible for the function of MIR125B1 involved in autophagy. Importantly, the knockdown phenotypes for DRAM2 are similar to the effects of overexpressing MIR125B1 as impairment of PML-RARA degradation, inhibition of autophagy, and myeloid cell differentiation arrest. These effects of MIR125B1 and its target DRAM2 were further confirmed in an APL mouse model. Thus, MIR125B1 dysregulation may interfere with the effectiveness of ATRA-mediated differentiation through an autophagy-dependent pathway, representing a novel potential APL therapeutic target.     

MIR125B1 represses the degradation of the PML-RARA oncoprotein by an autophagy-lysosomal pathway in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)-associated PML-RARA fusion gene. We have previously found that MIR125B1 is highly expressed in patients with APL and may be associated with disease pathogenesis; however, the mechanism by which MIR125B1 exerts its oncogenic potential has not been fully elucidated. Here, we demonstrated that MIR125B1 abundance correlates with the PML-RARA status. MIR125B1 overexpression enhanced PML-RARA expression and inhibited the ATRA-induced degradation of the PML-RARA oncoprotein. RNA-seq analysis revealed a direct link between the PML-RARA degradation pathway and MIR125B1-arrested differentiation. We further demonstrated that the MIR125B1-mediated blockade of PML-RARA proteolysis was regulated via an autophagy-lysosomal pathway, contributing to the inhibition of APL differentiation. Furthermore, we identified DRAM2 (DNA-damage regulated autophagy modulator 2), a critical regulator of autophagy, as a novel target that was at least partly responsible for the function of MIR125B1 involved in autophagy. Importantly, the knockdown phenotypes for DRAM2 are similar to the effects of overexpressing MIR125B1 as impairment of PML-RARA degradation, inhibition of autophagy, and myeloid cell differentiation arrest. These effects of MIR125B1 and its target DRAM2 were further confirmed in an APL mouse model. Thus, MIR125B1 dysregulation may interfere with the effectiveness of ATRA-mediated differentiation through an autophagy-dependent pathway, representing a novel potential APL therapeutic target.     

A novel family of membrane-bound E3 ubiquitin ligases

A novel E3 ubiquitin ligase family that consists of viral E3 ubiquitin ligases (E3s) and their mammalian homologues was recently discovered. These novel E3s are membrane-bound molecules that share the secondary structure and catalytic domain for E3 activity. All family members have two transmembrane regions at the center and a RING-CH domain at the amino terminus. Forced expression of these novel E3s has been shown to reduce the surface expression of various membrane proteins through ubiquitination of target molecules. Initial examples of viral E3s were identified in Kaposi's sarcoma associated herpesvirus (KSHV) and murine gamma-herpesvirus 68 (MHV-68) and have been designated as modulator of immune recognition (MIR) 1, 2 and mK3, respectively. MIR 1, 2 and mK3 are able to down-regulate MHC class I molecule expression, and mK3 is required to establish an effective latent viral infection in vivo. The first characterized mammalian homologue to MIR 1, 2 and mK3 is c-MIR/MARCH VIII. Forced expression of c-MIR/MARCH VIII down-regulates B7-2, a co-stimulatory molecule important for antigen presentation. Subsequently, several mammalian molecules related to c-MIR/MARCH VIII have been characterized and named as membrane associated RING-CH (MARCH) family. However, the precise physiological function of MARCH family members remains as yet unknown.     

哺乳动物DMRT1基因调控区分析

dmrt1基因是迄今为止发现的第一个在种属间具有进化保守性的性别分化基因。该基因在哺乳动物的性别分化过程中发挥着重要作用。通过对哺乳动物dmrt1基因5’端和3’端调控区的分析,发现它们分别存在3个和7个同源性较高（＞ 60％）的保守区。PCR扩增得到该基因的启动子、3’端调控区及编码区,并构入表达载体转染COS-7和ST细胞,结果显示,克隆的5’端和3’端调控区都能有效引导报告基因gfp以及dmrt1基因的表达,但表达效率在不同细胞中存在较大差异,表明dmrt1的表达存在着细胞特异性及复杂的调控机制。     

Thioredoxins of a parasitic nematode: comparison of the 16- and 12-kDA thioredoxins from Haemonchus contortus

Thioredoxins are a family of small proteins conserved through evolution, which are essential for the maintenance of cellular homeostasis. The "classic" thioredoxin, identified in most species, is a 12-kDa protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in nematodes a larger protein, 16 kDa, with a Cys-Pro-Pro-Cys (CPPC) active site was identified. We report that in the parasitic nematode Haemonchus contortus, both the 12-kDa (HcTrx1) and the 16-kDa (HcTrx3) species are expressed through the life cycle. However, the HcTrx3 is expressed at higher concentrations. Recombinant HcTrx1 and HcTrx3 were produced and both reduced insulin at a rate similar to that observed with ovine (host) and Escherichia coli thioredoxins and both were regenerated by a mammalian thioredoxin reductase, demonstrating that they have similar thioredoxin activity. Unlike mammalian thioredoxins, both proteins were able to reduce oxidised glutathione and hydrogen peroxide. This suggests essential roles for these proteins in response to oxidative stress and the host immune attack. Analysis of ivermectin-resistant H. contortus showed that expression of both genes were increased in a drug-resistant strain relative to a sensitive strain. Involvement in drug resistance identifies these thioredoxin proteins as potential drug targets for parasite control.     

The R2TP complex: discovery and functions

The two closely related AAA+family ATPases Rvb1 and Rvb2 are part of several critical multiprotein complexes, and, thus, are involved in a wide range of cellular processes including chromatin remodelling, telomerase assembly, and snoRNP biogenesis. It was found that Rvb1 and Rvb2 form a tight functional complex with Pih1 (Protein interacting with Hsp90) and Tah1 (TPR-containing protein associated with Hsp90), which are two Hsp90 interactors. We named the complex R2TP. The complex was originally isolated from Saccharomyces cerevisiae and was, subsequently, identified in mammalian cells. R2TP was found to be required for box C/D snoRNP biogenesis in yeast and mammalian cells. More recently, several studies revealed that the complex is also involved in multiple biological processes including apoptosis, phosphatidylinositol-3 kinase-related protein kinase (PIKK) signalling, and RNA polymerase II assembly. In this review, we describe the discovery of the complex and discuss the emerging critical roles that R2TP plays in distinct cellular processes.     

Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity

AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.     

Protein networks reveal organ-specific defense strategies in maize in response to an aboveground herbivore

Many of the proteins and defense pathways in maize that are activated in an organ-specific manner in leaves and roots during aboveground caterpillar attack have not yet been identified. In this study, we examined systemic and organ-specific defenses in the insect-resistant maize genotype, Mp708, when infested aboveground with fall armyworm (FAW, Spodoptera frugiperda). We used proteomic and network biology analyses and then integrated these data with known FAW resistance QTL to create a protein abundance QTL (pQTL) subnetwork. Using 10-plex tandem mass spectrometry tags (TMT) proteomics technique, we identified a total of 4675 proteins in leaves and roots of control and FAW-infested plants. Among the identified proteins, 794 had significant differences in abundance in response to FAW herbivory. Proteins that were upregulated in leaves during FAW infestation included jasmonic acid biosynthetic enzymes, cysteine proteases, protease inhibitors, REDOX-related proteins, and peroxidases. In roots, highly abundant proteins were involved in ET biosynthesis, DNA expression regulation, and pyruvate biosynthesis. We found many proteins that possibly contribute different defense functions to FAW resistance in Mp708. One potential resistance mechanism identified was that trade-offs between growth and defense responses were reduced in Mp708. Some of the proteins involved in this trade-off that were found within the pQTL subnetwork were the Kinesin-like protein (GRMZM2G046186_P01) and Pi starvation-induced protein (GRMZM2G118037_P01). We proposed other mechanisms contributing to resistance that suggest that jasmonic acid and ethylene control the local accumulation of insecticidal cysteine protease (MIR1-CP) in leaves, while ethylene controlled the systemic accumulation of MIR1-CP in roots. Finally, we hypothesized that receptor kinases such as receptor protein kinase 1 (GRMZM2G055678) could be involved in the activation of root-specific defense responses during aboveground insect infestation.