Pull-down Approaches Research Articles

CircARAP2 controls sMICA-induced NK cell desensitization by erasing CTCF/PRC2-induced suppression in early endosome marker RAB5A

Natural killer cells (NK) are the "professional killer" of tumors and play a crucial role in anti-tumor immunotherapy. NK cell desensitization is a key mechanism of tumor immune escape. Dysregulated NKG2D-NKG2DL signaling is a primary driver of this desensitization process. However, the factors that regulate NK cell desensitization remain largely uncharacterized. Here, we present the first report that circular RNA circARAP2 (hsa_circ_0069396) is involved in the soluble MICA (sMICA)-induced NKG2D endocytosis in the NK cell desensitization model. CircARAP2 was upregulated during NK cell desensitization and the loss of circARAP2 alleviated NKG2D endocytosis and NK cell desensitization. Using Chromatin isolation by RNA purification (ChIRP) and RNA pull-down approaches, we identified that RAB5A, a molecular marker of early endosomes, was its downstream target. Notably, transcription factor CTCF was an intermediate functional partner of circARAP2. Mechanistically, we discovered that circARAP2 interacted with CTCF and inhibited the recruitment of CTCF-Polycomb Repressive Complex 2 (PRC2) to the promoter region of RAB5A, thereby erasing histone H3K27 and H3K9 methylation suppression to enhance RAB5A transcription. These data demonstrate that inhibition of circARAP2 effectively alleviates sMICA-induced NKG2D endocytosis and NK cell desensitization, providing a novel target for therapeutic intervention in tumor immune evasion.

CpSmt3, an ortholog of small ubiquitin-like modifier, is essential for growth, organelle function, virulence, and antiviral defense in Cryphonectria parasitica.

SUMOylation is an important post-translational modification that regulates the expression, localization, and activity of substrate proteins, thereby participating in various important cellular processes such as the cell cycle, cell metabolism, gene transcription, and antiviral activity. However, the function of SUMOylation in phytopathogenic fungi has not yet been adequately explored. A comprehensive analysis composed of proteomics, affinity pull-down, molecular and cellular approaches was performed to explore the roles of SUMOylation in Cryphonectria parasitica, the fungal pathogen responsible for chestnut blight. CpSmt3, the gene encoding the SUMO protein CpSmt3 in C. parasitica was identified and characterized. Deletion of the CpSmt3 gene resulted in defects in mycelial growth and hyphal morphology, suppression of sporulation, attenuation of virulence, weakening of stress tolerance, and elevated accumulation of hypovirus dsRNA. The ΔCpSmt3 deletion mutant exhibited an increase in mitochondrial ROS, swollen mitochondria, excess autophagy, and thickened cell walls. About 500 putative SUMO substrate proteins were identified by affinity pull-down, among which many were implicated in the cell cycle, ribosome, translation, and virulence. Proteomics and SUMO substrate analyses further revealed that deletion of CpSmt3 reduced the accumulation of CpRho1, an important protein that is involved in TOR signal transduction. Silencing of CpRho1 resulted in a phenotype similar to that of ΔCpSmt3, while overexpression of CpRho1 could partly rescue some of the prominent defects in ΔCpSmt3. Together, these findings demonstrate that SUMOylation by CpSmt3 is vitally important and provide new insights into the SUMOylation-related regulatory mechanisms in C. parasitica.

Stability-Based Proteomics for Investigation of Structured RNA-Protein Interactions.

RNA-protein interactions are essential to RNA function throughout biology. Identifying the protein interactions associated with a specific RNA, however, is currently hindered by the need for RNA labeling or costly tiling-based approaches. Conventional strategies, which commonly rely on affinity pull-down approaches, are also skewed to the detection of high affinity interactions and frequently miss weaker interactions that may be biologically important. Reported here is the first adaptation of stability-based mass spectrometry methods for the global analysis of RNA-protein interactions. The stability of proteins from rates of oxidation (SPROX) and thermal protein profiling (TPP) methods are used to identify the protein targets of three RNA ligands, the MALAT1 triple helix (TH), a viral stem loop (SL), and an unstructured RNA (PolyU), in LNCaP nuclear lysate. The 315 protein hits with RNA-induced conformational and stability changes detected by TPP and/or SPROX were enriched in previously annotated RNA-binding proteins and included new proteins for hypothesis generation. Also demonstrated are the orthogonality of the SPROX and TPP approaches and the utility of the domain-specific information available with SPROX. This work establishes a novel platform for the global discovery and interrogation of RNA-protein interactions that is generalizable to numerous biological contexts and RNA targets.

DNALI1 interacts with the MEIG1/PACRG complex within the manchette and is required for proper sperm flagellum assembly in mice.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

The Role of GmSnRK1-GmNodH Module in Regulating Soybean Nodulation Capacity.

SnRK1 protein kinase plays hub roles in plant carbon and nitrogen metabolism. However, the function of SnRK1 in legume nodulation and symbiotic nitrogen fixation is still elusive. In this study, we identified GmNodH, a putative sulfotransferase, as an interacting protein of GmSnRK1 by yeast two-hybrid screen. The qRT-PCR assays indicate that GmNodH gene is highly expressed in soybean roots and could be induced by rhizobial infection and nitrate stress. Fluorescence microscopic analyses showed that GmNodH was colocalized with GsSnRK1 on plasma membrane. The physical interaction between GmNodH and GmSnRK1 was further verified by using split-luciferase complementary assay and pull-down approaches. In vitro phosphorylation assay showed that GmSnRK1 could phosphorylate GmNodH at Ser193. To dissect the function and genetic relationship of GmSnRK1 and GmNodH in soybean, we co-expressed the wild-type and mutated GmSnRK1 and GmNodH genes in soybean hairy roots and found that co-expression of GmSnRK1/GmNodH genes significantly promoted soybean nodulation rates and the expression levels of nodulation-related GmNF5α and GmNSP1 genes. Taken together, this study provides the first biological evidence that GmSnRK1 may interact with and phosphorylate GmNodH to synergistically regulate soybean nodulation.

Cell surface biotinylation to identify the receptors involved in nanoparticle uptake into endothelial cells

Targeted drug delivery requires –among others- specific interaction of nanocarriers with cell surface receptors enabling efficient internalization into the targeted cells. Thus, identification of receptors allowing efficient nanocarrier uptake is essential to improve the design of targeted nanomedicines. Here we used methods based on cell surface biotinylation to identify cell surface receptors mediating nanoparticle uptake by cells. We used human brain and liver endothelial cells as representative examples of cells typically showing very low and very high nanoparticle uptake, respectively. Amino-modified and carboxylated silica were used as model nanoparticles usually associated with high and low uptake into cells, respectively, and carrying different coronas after exposure in full human plasma. Using cell surface biotinylation of live cells and receptor pull-down assays, we compared the receptors internalized in control untreated cells and those internalized upon exposure to nanoparticles. In this way, we identified receptors associated with (high) nanoparticle uptake. The candidate receptors were further validated by decorating the nanoparticles with an artificial corona consisting of the respective receptor ligands. We found that a vitronectin corona can be used to target integrin receptors and strongly enhances nanoparticle uptake in brain and liver endothelial cells. The increased uptake was maintained in the presence of serum, suggesting that the vitronectin-corona could resist interaction and competition with serum. Furthermore, plasminogen-coated nanoparticles promoted uptake in endothelial cells of the liver, but not of the brain. The presented approach using reversible biotinylation of cell surface receptors in live cells allows for receptor-based targeting of nanocarriers that are instrumental in nanoparticle uptake, which can be exploited for targeted drug delivery. Statement of significanceIn order to deliver drugs to their site of action, drug-loaded nanocarriers can be targeted to cell receptors enabling efficient uptake into target cells. Thus, methods to identify nanocarrier receptors are invaluable. Here we used reversible biotinylation of live cells and receptor pull-down approaches for receptor identification. By comparative analysis of the individual receptors internalized in untreated cells and cells exposed to nanoparticles, we identified receptors enabling high nanoparticle uptake into liver and brain endothelial cells. Their role was confirmed by decorating nanoparticles with an artificial corona composed of the receptor ligands. In conclusion, live cell reversible biotinylation of cell surface proteins is a powerful tool for the identification of potential receptors for receptor-based targeting of nanocarriers.

A Photo-Crosslinking Approach to Identify Class II SUMO-1 Binders.

The small ubiquitin-like modifier (SUMO) is involved in various cellular processes and mediates known non-covalent protein-protein interactions by three distinct binding surfaces, whose interactions are termed class I to class III. While interactors for the class I interaction, which involves binding of a SUMO-interacting motif (SIM) to a hydrophobic groove in SUMO-1 and SUMO-2/3, are widely abundant, only a couple of examples have been reported for the other two types of interactions. Class II binding is conveyed by the E67 loop region on SUMO-1. Many previous studies to identify SUMO binders using pull-down or microarray approaches did not strategize on the SUMO binding mode. Identification of SUMO binding partners is further complicated due to the typically transient and low affinity interactions with the modifier. Here we aimed to identify SUMO-1 binders selectively enriched for class II binding. Using a genetically encoded photo-crosslinker approach, we have designed SUMO-1 probes to covalently capture class II SUMO-1 interactors by strategically positioning the photo-crosslinking moiety on the SUMO-1 surface. The probes were validated using known class II and class I binding partners. We utilized the probe with p-benzoyl-phenylalanine (BzF, also termed BpF or Bpa) at the position of Gln69 to identify binding proteins from mammalian cell extracts using mass spectrometry. By comparison with results obtained with a similarly designed SUMO-1 probe to target SIM-mediated binders of the class I type, we identified 192 and 96 proteins specifically enriched by either probe, respectively. The implicated preferential class I or class II binding modes of these proteins will further contribute to unveiling the complex interplay of SUMO-1-mediated interactions.

Grad-seq identifies KhpB as a global RNA-binding protein in Clostridioides difficile that regulates toxin production.

Much of our current knowledge about cellular RNA-protein complexes in bacteria is derived from analyses in gram-negative model organisms, with the discovery of RNA-binding proteins (RBPs) generally lagging behind in Gram-positive species. Here, we have applied Grad-seq analysis of native RNA-protein complexes to a major Gram-positive human pathogen, Clostridioides difficile, whose RNA biology remains largely unexplored. Our analysis resolves in-gradient distributions for ∼88% of all annotated transcripts and ∼50% of all proteins, thereby providing a comprehensive resource for the discovery of RNA-protein and protein-protein complexes in C. difficile and related microbes. The sedimentation profiles together with pulldown approaches identify KhpB, previously identified in Streptococcus pneumoniae, as an uncharacterized, pervasive RBP in C. difficile. Global RIP-seq analysis establishes a large suite of mRNA and small RNA targets of KhpB, similar to the scope of the Hfq targetome in C. difficile. The KhpB-bound transcripts include several functionally related mRNAs encoding virulence-associated metabolic pathways and toxin A whose transcript levels are observed to be increased in a khpB deletion strain. Moreover, the production of toxin protein is also increased upon khpB deletion. In summary, this study expands our knowledge of cellular RNA protein interactions in C. difficile and supports the emerging view that KhpB homologues constitute a new class of globally acting RBPs in Gram-positive bacteria.

A non-flagellated biocontrol bacterium employs a PilZ-PilB complex to provoke twitching motility associated with its predation behavior

Lysobacter enzymogenes OH11 is a non-flagellated, ubiquitous soil bacterium with broad-spectrum antifungal activities. Although lacking flagella, it employs another type of motile behavior, known as twitching motility that is powered by type IV pilus (T4P) to move towards neighboring crop fungal pathogens to kill them as food. At present, little is known about how this non-flagellated bacterium controls twitching motility that is crucial for its predatory lifestyle. Herein, we present a report on how a non-canonical PilZ domain, PilZLe3639, controls such motility in the non-flagellated L. enzymogenes; it failed to bind with c-di-GMP but seemed to be required for twitching motility. Using bacterial two-hybrid and pull-down approaches, we identified PilBLe0708, one of the PilZLe3639-binding proteins that are essential for the bacterial twitching motility, could serve as an ATPase to supply energy for T4P extension. Through site-mutagenesis approaches, we identified one essential residue of PilZLe3639 that is required for its binding affinity with PilBLe0708 and its regulatory function. Besides, two critical residues within the ATPase catalytic domains of PilBLe0708 were detected to be essential for regulating twitching behavior but not involved in binding with PilZLe3639. Overall, we illustrated that the PilZ-PilB complex formation is indispensable for twitching motility in a non-flagellated bacterium.

The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein–protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311. Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro. In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.

Krüppel homologue 1 interacts directly with Hairy and regulates ecdysis in the brown planthopper.

Juvenile hormone (JH) plays important roles in the growth and development of insects. JH and its receptor methoprene-tolerant (Met) regulate the expression of transcription factors to control the transcription of downstream genes. The expression of Hairy (Hry) and Krüppel homologue 1 (Kr-h1) is regulated by JH and JH receptors. Hry and Kr-h1 are both crucial in mediating JH signalling. However, whether they interact at the gene level in regulating metamorphosis and whether they interact physically at the protein level remain unknown. We used co-immunoprecipitation, glutathione S-transferase pull-down and RNA interference (RNAi) approaches to study the genetic and biochemical interactions of the two proteins Hry and Kr-h1. The results showed that brown planthopper (Nilaparvata lugens) Hry and Kr-h1 interact directly: Hry binds to the N-terminal of Kr-h1, which includes five zinc-finger domains. The RNAi experiment showed that downregulation of Hry reduced the ratio of ecdysis failure caused by knockdown of Kr-h1, indicating that the downregulation of Hry might mitigate ecdysis failure via the downregulation of Kr-h1. The expression of Hry increased significantly when Kr-h1 was downregulated, whereas it did not change significantly when both were downregulated. Our results suggest that the binding of Hry protein with Kr-h1 prevents the N-terminal five zinc-finger domains from binding with DNA, which in turn inactivates the transcription activator or inhibitor function of Kr-h1. Hry could possibly be used as a target for pesticide applications in the future.

Investigation of Novel Cavin-1/Suppressor of CytokineSignaling 3 (SOCS3) Interactions by Coimmunoprecipitation, Peptide Pull-Down, and Peptide Array Overlay Approaches.

The ability of inducible regulator suppressor of cytokine signaling 3 (SOCS3) to inhibit Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling requires interaction with specific cytokine receptors, JAKs, and components of the cellular ubiquitylation machinery. However, it is now clear that additional protein interactions are essential for effective inhibition of JAK-STAT signaling that have also identified new roles for SOCS3. For example, we have demonstrated that SOCS3 interaction with cavin-1, a core component of caveolae essential for their formation, is required for effective inhibition of interleukin (IL)-6 signaling and maintenance of cellular levels of caveolae. This is achieved through cavin-1 interaction with a discrete motif within the SOCS3 SH2 domain. Here, we describe in detail three methods (coimmunoprecipitation; peptide pull-down; peptide array overlay) we have used to validate and characterize cavin-1/SOCS3 interactions in vitro.

ErbB3-binding protein 1 (EBP1) represses HNF4α-mediated transcription and insulin secretion in pancreatic β-cells

HNF4α (hepatocyte nuclear factor 4α) is one of the master regulators of pancreatic β-cell development and function, and mutations in the HNF4α gene are well-known monogenic causes of diabetes. As a member of the nuclear receptor family, HNF4α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge of the different functional complexes in which HNF4α participates. Here, to find HNF4α-binding proteins in pancreatic β-cells, we used yeast two-hybrid screening, a mammalian two-hybrid assay, and glutathione S-transferase pulldown approaches, which identified EBP1 (ErbB3-binding protein 1) as a factor that binds HNF4α in a LXXLL motif-mediated manner. In the β-cells, EBP1 suppressed the expression of HNF4α target genes that are implicated in insulin secretion, which is impaired in HNF4α mutation-driven diabetes. The crystal structure of the HNF4α ligand-binding domain in complex with a peptide harboring the EBP1 LXXLL motif at 3.15Å resolution hinted at the molecular basis of the repression. The details of the structure suggested that EBP1's LXXLL motif competes with HNF4α coactivators for the same binding pocket and thereby prevents recruitment of additional transcriptional coactivators. These findings provide further evidence that EBP1 plays multiple cellular roles and is involved in nuclear receptor-mediated gene regulation. Selective disruption of the HNF4α-EBP1 interaction or tissue-specific EBP1 inactivation can enhance HNF4α activities and thereby improve insulin secretion in β-cells, potentially representing a new strategy for managing diabetes and related metabolic disorders.

A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

Porcine circovirus type 2 (PCV2) is the infectious agent of postweaning multisystemic wasting syndrome (PMWS). The recently discovered open reading frame 5 (ORF5) in PCV2 genome encodes a non-structural protein. Previous study revealed that ORF5 protein inhibits cell proliferation and may interact with host transmembrane glycoprotein NMB (GPNMB). However, whether the GPNMB affects PCV2 replication and the underlying molecular mechanisms are still unknown. In this study, the transcriptome maps of PCV2-infected and ORF5-transfected porcine alveolar macrophages 3D4/2 (PAM) cells were profiled. The GPNMB gene was down-regulated in PCV2-infected and ORF5-transfected PAMs. By using glutathione S-transferase (GST) pull-down, co-immunoprecipitation (co-IP) and confocal microscopy approaches, we convincingly showed that PCV2 ORF5 protein interacts with GPNMB. Furthermore, by utilizing lentivirus mediated overexpression or knockdown approach, we showed that the cellular GPNMB significantly inhibits PCV2 replication and ORF5 expression. Moreover, GPNMB overexpressing leads to an increased Cyclin A expression and a reduced S phase, whereas GPNMB knockdown causes a decreased Cyclin A expression and a prolonged S phase. In conclusion, we identified a novel host factor GPNMB that interacts with PCV2 ORF5 protein and restricts PCV2 replication.

APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting.

RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase-activating proteins (GAPs). Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull-down or yeast two-hybrid approaches, have identified a number of RAB interactors. However, due to the invitro nature of these approaches and inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2-mediated proximity labeling of RAB4a, RAB5a, RAB7a, and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2-mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.

Nucleotide exchange factor Rab3GEP requires DENN and non-DENN elements for activation and targeting of Rab27a.

ABSTRACTRab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3GKO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3GKO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.

The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation–mediated 14-3-3 binding

The tuberous sclerosis complex (TSC) is a negative regulator of mTOR complex 1, a signaling node promoting cellular growth in response to various nutrients and growth factors. However, several regulators in TSC signaling still await discovery and characterization. Using pulldown and MS approaches, here we identified the TSC complex member, TBC1 domain family member 7 (TBC1D7), as a binding partner for PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), a negative regulator of Akt kinase signaling. Most TBC domain-containing proteins function as Rab GTPase-activating proteins (RabGAPs), but the crystal structure of TBC1D7 revealed that it lacks residues critical for RabGAP activity. Sequence analysis identified a putative site for both Akt-mediated phosphorylation and 14-3-3 binding at Ser-124, and we found that Akt phosphorylates TBC1D7 at Ser-124. However, this phosphorylation had no effect on the binding of TBC1D7 to TSC1, but stabilized TBC1D7. Moreover, 14-3-3 protein both bound and stabilized TBC1D7 in a growth factor-dependent manner, and a phospho-deficient substitution, S124A, prevented this interaction. The crystal structure of 14-3-3ζ in complex with a phospho-Ser-124 TBC1D7 peptide confirmed the direct interaction between 14-3-3 and TBC1D7. The sequence immediately upstream of Ser-124 aligned with a canonical β-TrCP degron, and we found that the E3 ubiquitin ligase β-TrCP2 ubiquitinates TBC1D7 and decreases its stability. Our findings reveal that Akt activity determines the phosphorylation status of TBC1D7 at the phospho-switch Ser-124, which governs binding to either 14-3-3 or β-TrCP2, resulting in increased or decreased stability of TBC1D7, respectively.

Abstract 3033: The PRL family of tyrosine phosphatases as a novel drug targets in acute lymphoblastic leukemia

Abstract The aggressive and unpredictable behavior of acute lymphoblastic leukemia (ALL) presents a major clinical challenge in both the pediatric and adult setting. The development of new and better chemotherapies for this disease requires a detailed understanding of the genes and pathways that drive ALL malignancy. Phosphatases are emerging as new drug targets with important roles in leukemia progression. To determine the extent to which phosphatases play a role in ALL malignancy, we used CRISPR/Cas9 to knock out expression of 255 individual phosphatases in human T-cell acute lymphoblastic leukemia cell lines and screened for deleterious effects on cell viability. Top hits from this screen included the protein tyrosine phosphatase type IVa family, also known as the PRLs. PRL3 is genomically amplified in a subset of human T-ALL, and more than 50% of human ALL patients have significantly higher expression of PRL2 and/or PRL3, compared to normal lymphocytes, suggesting that these PRLs may play an important role in ALL malignancy. Transgenic Myc-induced ALL models in zebrafish showed that overexpression of PRL2 and PRL3 significantly shortened latency of primary and relapse ALL, enhanced leukemia stem cell self-renewal (PRL2) and prevented apoptosis after standard chemotherapy treatment (PRL3). Finally, a specific PRL inhibitor strongly induced apoptosis of PRL-expressing ALL cells in a dose-dependent manner, in both zebrafish models in vivo and human cell lines in vitro. We have identified several FDA-approved, general phosphatase inhibitors that have potent anti-PRL activity and are capable of killing ALL cells in vitro. Current work is focused on moving these inhibitors into pre-clinical testing using patient-derived xenografts. Pull-down approaches are also being used to identify the substrates of PRL phosphatase activity that are critical to ALL survival and may represent new, tractable drug targets for the treatment of ALL. <!–EndFragment–> Citation Format: Jessica S. Blackburn. The PRL family of tyrosine phosphatases as a novel drug targets in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3033. doi:10.1158/1538-7445.AM2017-3033

MiR-219 Cooperates with miR-338 in Myelination and Promotes Myelin Repair in the CNS

A lack of sufficient oligodendrocyte myelination contributes to remyelination failure in demyelinating disorders. miRNAs have been implicated in oligodendrogenesis; however, their functions in myelin regeneration remained elusive. Through developmentally regulated targeted mutagenesis, we demonstrate that miR-219 alleles are critical for CNS myelination and remyelination after injury. Further deletion of miR-338 exacerbates the miR-219 mutant hypomyelination phenotype. Conversely, miR-219 overexpression promotes precocious oligodendrocyte maturation and regeneration processes in transgenic mice. Integrated transcriptome profiling and biotin-affinity miRNA pull-down approaches reveal stage-specific miR-219 targets in oligodendrocytes and further uncover a novel network for miR-219 targeting of differentiation inhibitors including Lingo1 and Etv5. Inhibition of Lingo1 and Etv5 partially rescues differentiation defects of miR-219-deficient oligodendrocyte precursors. Furthermore, miR-219 mimics enhance myelin restoration following lysolecithin-induced demyelination as well as experimental autoimmune encephalomyelitis, principal animal models of multiple sclerosis. Together, our findings identify context-specific miRNA-regulated checkpoints that control myelinogenesis and a therapeutic role for miR-219 in CNS myelin repair.

Interactome of E. piscicida and grouper liver proteins reveals strategies of bacterial infection and host immune response

The occurrence of infectious diseases is related to heterogeneous protein interactions between a host and a microbe. Therefore, elucidating the host-pathogen interplay is essential. We previously revealed the protein interactome between Edwardsiella piscicida and fish gill cells, and the present study identified the protein interactome between E. piscicida and E. drummondhayi liver cells. E. drummondhayi liver cells and bacterial pull-down approaches were used to identify E. piscicida outer membrane proteins that bind to liver cells and fish liver cell proteins that interact with bacterial cells, respectively. Eight bacterial proteins and 11 fish proteins were characterized. Heterogeneous protein-protein interactions between these bacterial cells and fish liver cells were investigated through far-Western blotting and co-immunoprecipitation. A network was constructed based on 42 heterogeneous protein-protein interactions between seven bacterial proteins and 10 fish proteins. A comparison of the new interactome with the previously reported interactome showed that four bacterial proteins overlapped, whereas all of the identified fish proteins were new, suggesting a difference between bacterial tricks for evading host immunity and the host strategy for combating bacterial infection. Furthermore, these bacterial proteins were found to regulate the expression of host innate immune-related proteins. These findings indicate that the interactome contributes to bacterial infection and host immunity.