Protein-protein Interaction Motifs Research Articles

Crystal structure of a plant leucine rich repeat protein with two island domains

Leucine rich repeat (LRR) domain, characterized by a repetitive sequence pattern rich in leucine residues, is a universal protein-protein interaction motif present in all life forms. LRR repeats interrupted by sequences of 30-70 residues (termed island domain, ID) have been found in some plant LRR receptor-like kinases (RLKs) and animal Toll-like receptors (TLR7-9). Recent studies provide insight into how a single ID is structurally integrated into an LRR protein. However, structural information on an LRR protein with two IDs is lacking. The receptor-like protein kinase 2 (RPK2) is an LRR-RLK and has important roles in controlling plant growth and development by perception and transduction of hormone signal. Here we present the crystal structure of the extracellular LRR domain of RPK2 (RPK2-LRR) containing two IDs from Arabidopsis. The structure reveals that both of the IDs are helical and located at the central region of the single RPK2-LRR solenoid. One of them binds to the inner surface of the solenoid, whereas the other one mainly interacts with the lateral side. Unexpectedly, a long loop immediately following the N-terminal capping domain of RPK2-LRR is presented toward and sandwiched between the two IDs, further stabilizing their embedding to the LRR solenoid. A potential ligand binding site formed by the two IDs and the solenoid is located at the C-terminal side of RPK2-LRR. The structural information of RPK2-LRR broadens our understanding toward the large family of LRR proteins and provides insight into RPK2-mediated signaling.

Stem Mutants in the N-terminal Domain of the Phage P22 Tailspike Protein

The P22 tailspike protein is an intensely studied protein whose structure and sequence has been described. However, a study, describing important protein interactions related to its function at the N-terminal domain, has been lacking. The P22 tailspike protein (TSP) consists of three identical polypeptide chains of 666aa. The first 108 of the 666aa in the P22 TSP form a trimeric N-terminal domain (NTD). Each of the three chains of the trimeric NTD contributes to the formation of a dome-like structure. Our studies suggest that a short stretch of amino acids located within the first fifteen amino acids of the P22 TSP NTD is critical for the stability of the dome structure formed by the first 108aa of the P22 TSP NTD. The first 23aa are located within this dome-like structure and have been dubbed the “stem” of the NTD. Although amino acid residues in the first 15aa (lower stem) are critical, deletion analysis and in vitro assembly studies implicate the rest of the stem in additional stabilizing interactions. Our studies implicate a common protein-protein interaction motif made up of interchain hydrophobic contacts between adjacent chains

Smarter neuronal signaling complexes from existing components: How regulatory modifications were acquired during animal evolution

Neurons of organisms with complex and flexible behavior, especially humans, must precisely control protein localization and activity to support higher brain functions such as learning and memory. In contrast, simpler organisms generally have simpler individual neurons, less complex nervous systems and display more limited behaviors. Strikingly, however, many key neuronal proteins are conserved between organisms that have very different degrees of behavioral complexity. Here we discuss a possible mechanism by which conserved neuronal proteins acquired new attributes that were crucial in the evolution of complexity of nervous system structure and function. Specifically, we hypothesize that vertebrate-specific post-translational palmitoylation sites and PDZ-binding protein-protein interaction motifs act as gain-of-function mutations, increasing the regulatory potential of conserved proteins without affecting their core functions. We further hypothesize that the additional regulation of neurotransmitter receptors and other membrane proteins made possible by these sites and motifs is critical for the function of complex nervous systems.

Regulation of TGFβ superfamily signaling by two separable domains of glypican LON-2 inC. elegans

Regulated intercellular signaling is critical for the normal development and maintenance of multicellular organisms. Glypicans have been shown to regulate signaling by TGFβs, hedgehogs and Wnts, in several cellular contexts. Glypicans comprise a conserved family of heparan sulfated, glycosylphosphatidylinositol (GPI)-linked extracellular proteins. The structural complexity of glypicans may underlie their functional complexity. In a recent study31, we built on previous findings that one of the two C. elegans glypicans, LON-2, specifically inhibits signaling by the TGFβ superfamily member DBL-1. We tested the functional requirements of LON-2 protein core components and post-translational modifications for LON-2 activity. We provide the first evidence that two parts of a glypican can independently regulate TGFβ superfamily signaling in vivo: the N-terminal furin protease product and a C-terminal region containing heparan sulfate attachment sites. Furthermore, we show a protein-protein interaction motif is crucial for LON-2 activity in the N-terminal protein core, suggesting that LON-2 acts by serving as a scaffold for DBL-1 and an RGD-binding protein. In addition, we demonstrate specificity of glypican function by showing C. elegans GPN-1 does not functionally substitute for LON-2. This work reveals a molecular foundation for understanding the complexity and specificity of glypican function.

Passing Messages between Biological Networks to Refine Predicted Interactions

Regulatory network reconstruction is a fundamental problem in computational biology. There are significant limitations to such reconstruction using individual datasets, and increasingly people attempt to construct networks using multiple, independent datasets obtained from complementary sources, but methods for this integration are lacking. We developed PANDA (Passing Attributes between Networks for Data Assimilation), a message-passing model using multiple sources of information to predict regulatory relationships, and used it to integrate protein-protein interaction, gene expression, and sequence motif data to reconstruct genome-wide, condition-specific regulatory networks in yeast as a model. The resulting networks were not only more accurate than those produced using individual data sets and other existing methods, but they also captured information regarding specific biological mechanisms and pathways that were missed using other methodologies. PANDA is scalable to higher eukaryotes, applicable to specific tissue or cell type data and conceptually generalizable to include a variety of regulatory, interaction, expression, and other genome-scale data. An implementation of the PANDA algorithm is available at www.sourceforge.net/projects/panda-net.

Functional Mapping of PilF and PilQ in the Pseudomonas aeruginosa Type IV Pilus System

Pseudomonas aeruginosa uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The P. aeruginosa T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein-protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from Pseudomonas cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined.

Identification of a Novel Protein-Protein Interaction Motif Mediating Interaction of GPCR-Associated Sorting Proteins with G Protein-Coupled Receptors

GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure–function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated “GASP motif” of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.

Ndc80 Loop as a protein-protein interaction motif

Our understanding of the structure and function of kinetochores has advanced dramatically over the past 10 years, yet how the plus end of spindle microtubules interacts with the kinetochore and establishes amphitelic attachment for proper sister chromatid segregation remains unresolved. However, several recent reports from different organisms have shed new light on this issue. A key player in microtubule-kinetochore interaction is the conserved Ndc80 outer kinetochore complex. In both yeast and human cells in particular, a ubiquitous internal ‘loop’ found in the Ndc80 molecule interrupting its C-terminal coiled-coil domain plays critical roles in protein-protein interaction, by recruiting microtubule-binding proteins to ensure proper kinetochore-microtubule attachment. In this commentary, we summarise the recent progress made and discuss the evolutionary significance of this loop’s role in microtubule dynamics at the kinetochore for accurate chromosome segregation.

The SOL-2/Neto Auxiliary Protein Modulates the Function of AMPA-Subtype Ionotropic Glutamate Receptors

The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

LST1/A Is a Myeloid Leukocyte-specific Transmembrane Adaptor Protein Recruiting Protein Tyrosine Phosphatases SHP-1 and SHP-2 to the Plasma Membrane

Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

BackgroundThe Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region.ResultsThe dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck.ConclusionBased on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

Roles of Mis18α in Epigenetic Regulation of Centromeric Chromatin and CENP-A Loading

The Mis18 complex has been identified as a critical factor for the centromeric localization of a histone H3 variant, centromeric protein A (CENP-A), which is responsible for the specification of centromere identity in the chromosome. However, the functional role of Mis18 complex is largely unknown. Here, we generated Mis18α conditional knockout mice and found that Mis18α deficiency resulted in lethality at early embryonic stage with severe defects in chromosome segregation caused by mislocalization of CENP-A. Further, we demonstrate Mis18α's crucial role for epigenetic regulation of centromeric chromatin by reinforcing centromeric localization of DNMT3A/3B. Mis18α interacts with DNMT3A/3B, and this interaction is critical for maintaining DNA methylation and hence regulating epigenetic states of centromeric chromatin. Mis18α deficiency led to reduced DNA methylation, altered histone modifications, and uncontrolled noncoding transcripts in centromere region by decreased DNMT3A/3B enrichment. Together, our findings uncover the functional mechanism of Mis18α and its pivotal role in mammalian cell cycle.

The adaptor protein p62/SQSTM1 in osteoclast signaling pathways

Paget's disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover and overactive osteoclasts. The discovery of mutations in the SQSTM1/p62 gene in numerous patients has identified protein p62 as an important modulator of bone turnover. In both precursors and mature osteoclasts, the interaction between receptor activator of NF-κB ligand (RANKL) and its receptor RANK results in signaling cascades that ultimately activate transcription factors, particularly NF-κB and NFATc1, promoting and regulating the osteoclast differentiation, activity, and survival. As a scaffold with multiple protein-protein interaction motifs, p62 is involved in virtually all the RANKL-activated osteoclast signaling pathways, along with being implicated in numerous other cellular processes. The p62 adaptor protein is one of the functional links reported between RANKL and TRAF6-mediated NF-κB activation, and also plays a major role as a shuttling factor that targets polyubiquitinated proteins for degradation by either the autophagy or proteasome pathways. The dysregulated expression and/or activity of p62 in bone disease up-regulates osteoclast functions. This review aims to outline and summarize the role of p62 in RANKL-induced signaling pathways and in ubiquitin-mediated signaling in osteoclasts, and the impact of PDB-associated p62 mutations on these processes.

Expressions and Roles of AMIGO Gene Family in Vascular Endothelial Cells

In this study, we have developed shRNA expression vector and determined their RNAi activity. The endothelial cells (ECs) were transfected with the Amphoterin-induced gene and ORF (AMIGO-2) expression vector or AMIGO-2 shRNA expression vector using hemagglutinating virus of Japan (HVJ) Envelope Vector and then the cells were assayed for AMIGO-2 mRNA levels and its survival. In our study, here we have performed expression of AMIGO gene family in primary cultured human vascular cells, and found predominant expression on human microvascular endothelial cells (HMVEC). The expression of AMIGO-2 gene in HMVEC under hypoxia, showed AMIGO-2 gene decreased significantly, suggested that AMIGO-2 maybe involved in vascular remodeling. Based on our study of AMIGO-2 down regulation and over expression showed the down regulation appeared to cause cell death of ECs, and over expression appeared to protect EC death due to reactive oxygen species. Finally, our this study suggest that AMIGO-2 may have an important role in the vascular system as a cell survival promoting factor for vascular ECs, probably as being involved in vascular development, angiogenesis and/or vascular remodeling. (where x can be any amino acid and L could also be replaced by V, I or F). The LRRs are protein-protein interaction motifs and are found in a large number of proteins (2). Some LRR-containing plasma membrane proteins are expressed almost exclusively in the brain, implying specific functions in the central nervous system. Expression analyses indicate that AMIGO-1 is specifically detected in axonal fibers and tracts in the brain. AMIGO-2 and AMIGO-3 expressions are more widespread but are also brain-enriched. Members of the AMIGO family exhibit both homophilic and heterophilic binding, which suggest that they function as novel cell adhesion molecules in neurons. Immobilized ectodomain of AMIGO-1 promoted neurite extension of cultured hippocampal neurons, but, when added to the medium, the same soluble AMIGO-1 inhibited fasciculation of neurites.

Molecular and Structural Mechanism of the Assembly of an Acid-Sensing Receptor/Ion Channel Complex

Proteins in the polycystic kidney disease (PKD) family associate with subunits of the transient receptor potential polysystin (TRPP) subfamily to form functionally important complexes. For example, PKD1 and TRPP2 form a receptor/ion channel complex critical for kidney development and function, and mutations in both proteins cause human polycystic kidney disease. Another PKD member, PKD1L3, coassemble with TRPP3 to form a candidate sour taste receptor. The molecular mechanisms of the assembly of these complexes are largely unknown. Unlike TRP channel subunits, which contain six transmembrane segments, PKD proteins have 11 putative transmembrane segments, with a large extracellular N-terminus containing well-recognized protein-protein, protein-sugar, and protein-ligand interaction motifs. PKD proteins are therefore generally believed to function as cell surface receptors. In this study, we find that PKD1L3, when expressed in Xenopus oocytes, functions as a channel-forming subunit in an acid-sensing heteromeric channel complex formed by PKD1L3 and TRPP3. Single amino acid mutations in the putative pore region of both proteins alter the channel's permeability to monovalent and divalent cations. Single-molecule imaging analysis indicates that the PKD1L3/TRPP3 complex in the plasma membrane of live Xenopus oocytes contains one PKD1L3 and three TRPP3 subunits. We identify a C-terminal coiled-coil domain in TRPP3 and find it to be a trimer in solution and crystal structure. This coiled-coil domain trimer is critical for the assembly and surface expression of TRPP3 homomeric complexes and PKD1L3/TRPP3 heteromeric complexes. These results reinforce the notion that PKD and TRPP proteins associate with a 1:3 stoichiometry. They also provide evidence demonstrating that PKD proteins can function as channel-forming subunits.

Nuclear Targeting of Methyl-Recycling Enzymes in Arabidopsis thaliana Is Mediated by Specific Protein Interactions

Numerous transmethylation reactions are required for normal plant growth and development. S-adenosylhomocysteine hydrolase (SAHH) and adenosine kinase (ADK) act coordinately to recycle the by-product of these reactions, S-adenosylhomocysteine (SAH) that would otherwise competitively inhibit methyltransferase (MT) activities. Here, we report on investigations to understand how the SAH produced in the nucleus is metabolized by SAHH and ADK. Localization analyses using green fluorescent fusion proteins demonstrated that both enzymes are capable of localizing to the cytoplasm and the nucleus, although no obvious nuclear localization signal was found in their sequences. Deletion analysis revealed that a 41-amino-acid segment of SAHH (Gly(150)-Lys(190)) is required for nuclear targeting of this enzyme. This segment is surface exposed, shows unique sequence conservation patterns in plant SAHHs, and possesses additional features of protein-protein interaction motifs. ADK and SAHH interact in Arabidopsis via this segment and also interact with an mRNA cap MT. We propose that the targeting of this complex is directed by the nuclear localization signal of the MT; other MTs may similarly target SAHH/ADK to other subcellular compartments to ensure uninterrupted transmethylation.

Abstract C177: Chemical-driven biological probing: Discovery of a novel antimetastatic drug.

Abstract Chemicals constitute richly informative probes, have high resolving potential and are able to uncover complex biological processes. Harnessing this potential provides an avenue for the discovery of new therapeutics that act via novel mechanisms. We focused these principles upon an intractable problem: cancer metastasis. We started with a chemical scaffold with broad bioactivity, with desirable drug-like properties and that would support synthetic diversification. Initially focusing upon human prostate cancer (PCa) we then coupled fragment-diversification and novel synthetic routes to upfront positive selection screens (inhibition of cell motility) and negative selection screens (cell toxicity) in an iterative fashion. We thereby efficiently synthesized a new class of bioactive compounds that inhibits systemic PCa metastasis at low nanomolar concentrations. Efficacy against other cancer types was demonstrated. Extensive investigations indicate high specificity and no toxicity. Target validation studies point to inhibition of protein-protein interaction motifs. Together, these studies support the notion that this approach is powerful, can be broadly applied across biological systems, and constitutes a paradigm. Specifically, they have led to the discovery of a novel acting drug that inhibits human cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C177.

CcmI Subunit of CcmFHI Heme Ligation Complex Functions as an Apocytochrome c Chaperone during c-Type Cytochrome Maturation

Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c(2) as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c(2) but not holocytochrome c(2). Mainly the C-terminal portions of both CcmI and apocytochrome c(2) mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c(2), like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c(2), but this interaction requires a free thiol group at apocytochrome c(2) heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae.

The ROXY1 C-terminal L**LL motif is essential for the interaction with TGA transcription factors.

Glutaredoxins (GRXs) are small, ubiquitous, glutathione-dependent oxidoreductases that participate in redox-regulated processes associated with stress responses. Recently, GRXs have been shown to exert crucial functions during flower developmental processes. GRXs modulate their target protein activities by the reduction of protein disulfide bonds or deglutathionylation reactions. The Arabidopsis (Arabidopsis thaliana) GRX ROXY1 participates in petal primordia initiation and further petal morphogenesis. ROXY1 belongs to a land plant-specific class of GRXs with a CC-type active site motif, deviating from the ubiquitously occurring CPYC and CGFS GRX classes. ROXY1 was previously shown to interact with floral TGA transcription factors in the nucleus, and this interaction is a prerequisite for ROXY1 to exert its activity required for Arabidopsis petal development. Deletion analysis further identified the importance of the ROXY1 C terminus for the ROXY1/TGA protein interactions and for the ROXY1 function in petal development. Here, by dissecting the ROXY1 C terminus, an α-helical L**LL motif immediately adjacent to the ROXY1 C-terminal eight amino acids was identified that is essential for the interaction with TGA transcription factors and crucial for the ROXY1 function in planta. Similar to the α-helical L**LL motifs binding to transcriptional coactivators with liganded nuclear receptors in animals, a hydrophobic face formed by the conserved leucines in the L**LL motif of ROXY1 possibly mediates the interaction with TGA transcription factors. Thus, the α-helical L**LL sequence is a conserved protein-protein interaction motif in both animals and plants. Furthermore, two separate TGA domains were identified by deletion experiments as being essential for mediating TGA protein interactions with ROXYs.

202 The Reduced and Altered Activities of PAX5 are Linked to the Protein-Protein Interaction Motif (Coiled-Coil Domain) of the PAX5-PML Fusion Protein in t(9;15)-Associated Acute Lymphocytic Leukemia

Abstract 1022 The paired box domain of PAX5 was reported to fuse with the sequence of promylocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia (B-ALL). In the present studies, we found, by gel-shift assays, that PAX5-PML bound to a panel of PAX5-consensus sequence as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction (RQ-PCR) analysis, PAX5-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(ΔCC) demonstrated that the coiled-coil protein interaction domain located within PML moiety was required for PAX5-PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP-PAX5-PML and YFP-PAX5-PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5-PML fusion protein has reduced intranuclear mobility compared to wild-type PAX5. Furthermore, the dimerization domain (coiled-coil) of PML was responsible for the reduced intranuclear mobility of PAX5-PML. These results indicate that the coiled-coil domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins. Disclosures: No relevant conflicts of interest to declare.