Protein-protein Interaction Motifs Research Articles

Abstract 2157: Cell cycle dependent regulation of c-Myb target gene specificity

Abstract The c-Myb transcription factor is a critical regulator of proliferation and stem cell differentiation, and mutated alleles of c-Myb are oncogenic, but little is known about changes in c-Myb activity during the cell cycle. Previous studies have shown that the specificity of c-Myb depends in part on its DNA binding domain, but also on protein-protein interaction motifs in the C-terminal regulatory domains of the protein. These interactions are likely subject to regulation by post-translational modifications and are modified in oncogenic derivatives of c-Myb, which regulate different sets of target genes than the normal c-Myb protein. The analysis of cell cycle regulated target genes provides an ideal means of following the regulation of c-Myb activity in different subsets of cells that are in distinct cell cycle phases. Using Chromatin Immunoprecipitation (ChIP) assays, we found that c-Myb associated with some target genes only in specific cell cycle stages. For example, with the Cyclin E1 gene promoter only in G2/M phase cells. However, we also found that the levels of c-Myb protein were dramatically affected by treating with some cell cycle blocking agents. To map the association of c-Myb with specific target genes during the cell cycle, we developed a novel Fix-Sort-ChIP approach, in which asynchronously growing cells were fixed with formaldehyde, stained with Hoechst 33342 and separated into different cell cycle fractions by flow sorting, then processed for ChIP assays. This approach showed that the specificity of c-Myb is dramatically different in small subpopulations of cells, for example cells in the G2/M phase of the cell cycle, than in the bulk population. The repositioning of c-Myb during the cell cycle is not due to changes in its expression and also occurs with ectopically expressed, epitope-tagged versions of c-Myb. The repositioning occurs in established cell lines, in primary human CD34+ hematopoietic progenitors and in primary human acute myeloid leukemia cells. Our results suggest that c-Myb specificity is actively regulated during the cell cycle, probably through changes in protein-protein interactions. This sheds new light on the dynamic nature of gene regulation during the cell cycle and on the importance of post-translational modifications and/or oncogenic mutations that can dramatically alter the activity and the specificity of transcription factors like c-Myb. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2157. doi:10.1158/1538-7445.AM2011-2157

Expression 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.

Photosynthetic characteristics of a multicellular green alga Volvox carteri in response to external CO2 levels possibly regulated by CCM1/CIA5 ortholog

When CO(2) supply is limited, aquatic photosynthetic organisms induce a CO(2)-concentrating mechanism (CCM) and acclimate to the CO(2)-limiting environment. Although the CCM is well studied in unicellular green algae such as Chlamydomonas reinhardtii, physiological aspects of the CCM and its associated genes in multicellular algae are poorly understood. In this study, by measuring photosynthetic affinity for CO(2), we present physiological data in support of a CCM in a multicellular green alga, Volvox carteri. The low-CO(2)-grown Volvox cells showed much higher affinity for inorganic carbon compared with high-CO(2)-grown cells. Addition of ethoxyzolamide, a membrane-permeable carbonic anhydrase inhibitor, to the culture remarkably reduced the photosynthetic affinity of low-CO(2) grown Volvox cells, indicating that an intracellular carbonic anhydrase contributed to the Volvox CCM. We also isolated a gene encoding a protein orthologous to CCM1/CIA5, a master regulator of the CCM in Chlamydomonas, from Volvox carteri. Volvox CCM1 encoded a protein with 701 amino acid residues showing 51.1% sequence identity with Chlamydomonas CCM1. Comparison of Volvox and Chlamydomonas CCM1 revealed a highly conserved N-terminal region containing zinc-binding amino acid residues, putative nuclear localization and export signals, and a C-terminal region containing a putative LXXLL protein-protein interaction motif. Based on these results, we discuss the physiological and genetic aspects of the CCM in Chlamydomonas and Volvox.

Sequence Motifs in MADS Transcription Factors Responsible for Specificity and Diversification of Protein-Protein Interaction

Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution.

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.

AbstractAbstract 1022The 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.

Identification of a variant form of tyrosine phosphatase LYP

BackgroundProtein tyrosine phosphatases (PTPs) are important cell signaling regulators with major pathological implications. LYP (also known as PTPN22) is an intracellular enzyme initially found to be predominately expressed in lymphocytes. Importantly, an allelic R620W variant of LYP is strongly associated with multiple autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and autoimmune thyroid disease.ResultsIn this study, we isolated a novel isoform of LYP designated LYP3. LYP3 differs from LYP1, the known isoform of LYP, in that it lacks a 28 amino acid segment right after the R620W site embedded in a proline-rich protein-protein interaction motif. Genomic sequence analysis revealed that LYP3 resulted from alternative splicing of the LYP gene located on chromosome 1p 13.3-13.1. Reverse transcription PCR analyses of 48 human tissues demonstrated that both LYP1 and LYP3 are predominantly expressed in primary and secondary lymphoid tissues but the relative expression levels of the two isoforms varies in different human tissues and individuals.ConclusionsWe thus identified a new variant form of LYP and conducted a comprehensive analysis of LYP tissue expressions. Considering the pathogenesis of LYP R620W, we believe that the expression of LYP3 may have an important role in regulating activity and function of LYP and may be implicated in autoimmune diseases.

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

Conservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity

Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.

Opposing Effects of a Tyrosine-Based Sorting Motif and a PDZ-Binding Motif Regulate Human T-Lymphotropic Virus Type 1 Envelope Trafficking

Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXPhi motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXPhi motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXPhi motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXPhi elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXPhi motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXPhi motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXPhi, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.

Biochemical and molecular features of LRRK2 and its pathophysiological roles in Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disease, and 5-10% of the PD cases are genetically inherited as familial PD (FPD). LRRK2 (leucine-rich repeat kinase 2) was first reported in 2004 as a gene corresponding to PARK8, an autosomal gene whose dominant mutations cause familial PD. LRRK2 contains both active kinase and GTPase domains as well as protein-protein interaction motifs such as LRR (leucine-rich repeat) and WD40. Most pathogenic LRRK2 mutations are located in either the GTPase or kinase domain, implying important roles for the enzymatic activities in PD pathogenic mechanisms. In comparison to other PD causative genes such as parkin and PINK1, LRRK2 exhibits two important features. One is that LRRK2's mutations (especially the G2019S mutation) were observed in sporadic as well as familial PD patients. Another is that, among the various PDcausing genes, pathological characteristics observed in patients carrying LRRK2 mutations are the most similar to patients with sporadic PD. Because of these two observations, LRRK2 has been intensively investigated for its pathogenic mechanism (s) and as a target gene for PD therapeutics. In this review, the general biochemical and molecular features of LRRK2, the recent results of LRRK2 studies and LRRK2's therapeutic potential as a PD target gene will be discussed.

Structural Basis of Dimerization-dependent Ubiquitination by the SCFFbx4 Ubiquitin Ligase

The F-box proteins are the substrate recognition subunits of the SCF (Skp1-Cul1-Rbx1-F- box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCF(Fbx4) (the superscript denotes the F-box protein) ubiquitination activity toward the telomere regulator Pin2 (also known as TRF1). The crystal structure of Fbx4 in complex with an adaptor protein Skp1 reveals an antiparallel dimer configuration in which the linker domain of Fbx4 interacts with the C-terminal substrate-binding domain of the other protomer, whereas the C-terminal domain of the protein adopts a compact alpha/beta fold distinct from those of known F-box proteins. Biochemical studies indicate that both the N-terminal domain and a loop connecting the linker and C-terminal domain of Fbx4 are critical for the dimerization and activation of the protein. Our findings provide a framework for understanding the role of F-box dimerization in the SCF-mediated ubiquitination reaction.

The structure of the mammalian RNase H2 complex provides insight into RNA:DNA hybrid processing to prevent immune dysfunction

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA:DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi‐Goutieres syndrome (AGS). We have determined the crystal structure of the three protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein‐protein interaction motifs that could allow for efficient participation in multiple cellular functions. Further structural and biochemical analyses show that some RNase H2 disease‐causing mutations likely result in aberrant protein‐protein interactions while the RNase H2A subunit‐G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification. Funding: NIH‐R01‐GM069962, Alliance for Lupus Research, ACS.

AlgK Is a TPR-Containing Protein and the Periplasmic Component of a Novel Exopolysaccharide Secretin

The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.

The Structure of the Mammalian RNase H2 Complex Provides Insight into RNA·DNA Hybrid Processing to Prevent Immune Dysfunction

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

Death‐associated protein kinase (DAPK) and signal transduction: additional roles beyond cell death

Death-associated protein kinase (DAPK) is a stress-regulated protein kinase that mediates a range of processes, including signal-induced cell death and autophagy. Although the kinase domain of DAPK has a range of substrates that mediate its signalling, the additional protein interaction domains of DAPK are relatively ill defined. This review will summarize our current knowledge of the DAPK interactome, the use of peptide aptamers to define novel protein-protein interaction motifs, and how these new protein-protein interactions give insight into DAPK functions in diverse cellular processes, including growth factor signalling, the regulation of autophagy, and its emerging role in the regulation of immune responses.

The Function of Yeast Epsin and Ede1 Ubiquitin‐Binding Domains During Receptor Internalization

The formation of a primary endocytic vesicle is a dynamic process involving the transient organization of adaptor and scaffold proteins at the plasma membrane. Epsins and Eps15-like proteins are ubiquitin-binding proteins that act early in this process. The yeast epsins, Ent1 and Ent2, carry functional ubiquitin-interacting motifs (UIMs), whereas the yeast Eps15-like protein, Ede1, has a C-terminal ubiquitin-associated (UBA) domain. Analysis of mutants lacking early endocytic adaptors reveals that the ubiquitin-binding domains (UBDs) of Ent2 and Ede1 are likely to function primarily to mediate protein-protein interactions between components of the early endocytic machinery. Cells that lack epsin and Ede1 UBDs are able to internalize activated, ubiquitinated receptors. Furthermore, under conditions in which epsin UIMs are important for receptor internalization, receptors internalized via both ubiquitin-dependent and ubiquitin-independent signals require the UIMs, indicating that UIM function is not restricted to ubiquitinated receptors. Epsin UIMs share function with non-UBD protein-protein interaction motifs in Ent2 and Ede1, and the Ede1 UBA domain appears to negatively regulate interactions between endocytic proteins. Together, our results suggest that the ubiquitin-binding domains within the yeast epsin Ent2 and Ede1 are involved in the formation and regulation of the endocytic network.

Abstract C63: Structure of the Skp1-Fbx4 complex reveals a new class of the SCF ubiquitin ligase

Abstract Ubiquitin-mediated proteolysis governs the levels of many cellular proteins via a triple-enzyme cascade, involving ubiquitin-activating (E1), conjugating (E2) and ligating (E3) activities. The E3 ligases recruit both E2 and specific substrates and catalyze the final step in ubiquitination reaction. SCF complexes are the largest family of E3 ubiquitin-protein ligases that consist of Cul1, Rbx1, Skp1 and a member of the F-box protein family. F-box proteins are the substrate-recognition components of SCF; they interact with Skp1 through the N-terminal ∼40-residue F box motif, to recruit substrates through their variable C-terminal protein-protein interaction domains, including WD40 repeats (Fbxw subfamily), leucine-rich repeats (LRRs; Fbxl subfamily) and other different or unknown domains (Fbxo subfamily). Fbx4, which is the F-box protein responsible for the recognition of cell-cycle regulator cyclin D1 and telomeric DNA-binding protein Pin2, belongs to the Fbxo family that lack recognizable protein-protein interaction motif in their C-terminus. Reduced Fbx4 expression appears to be responsible, at least in part, for overexpression of cyclin D1 in human cancers. On the other hand, overexpression of Fbx4 in human cells promotes degradation of endogenous Pin2 and results in progressive telomere elongation. In order to define the fundamental structural properties of Fbx4, we have determined the crystal structure of the human Fbx4 protein in complex with Skp1 by single-wavelength anomalous dispersion method. The structure of the binary complex reveals that Skp1 and the F-box domain of Fbx4 retain the same overall fold as in other Fbxl and Fbxw structures determined, and that binding of the F box to Skp1 is very similar to that in other Skp1-F box complexes. Interestingly, the C-terminal domain of Fbx4 is projected away from Skp1 and F box, to adopt an α/β fold with a seven-stranded β sheet surrounded by seven α helices, which is distinct from LRR (in Fbxl) and WD40 (in Fbxw), reinforcing the notion that Fbx4 represents a new family of substrate-recognition subunits in SCF E3s. Moreover, mutagenesis experiments have revealed a potential substrate-binding site in Fbx4 C-terminal domain. Taken together, our structural and biochemical data provide a framework for understanding the molecular mechanism of substrate recognition by the SCFFbx4 ubiquitin ligase. Citation Information: Cancer Res 2009;69(23 Suppl):C63.

Natural Genetic Engineering of Hepatitis C Virus NS5A for Immune System Counterattack

The Hepatitis C virus nonstructural 5A (NS5A) protein is a hydrophilic phosphoprotein with diverse functions. The domain assignment of NS5A had been refined using a systematic in silico bioinformatics approach using DOMAC, the protein is divided into three domains and domain III is subdivided into two subdomains using ProDom and SSEP servers. The fold structure for domains II and III were predicted using the meta-server 3D-Jury. Scanning motif databases (SMART, BLOCKS, and PROSITE) gave new motifs. Two important motifs, the interleukins 1 and 8 interaction motifs, relating to NS5A function in inducing the interleukin 8 promoter, were discovered from the BLOCKS scan. Protein-protein interaction motifs were predicted as hot loops and disordered regions, corresponding to binding regions with the ds-protein kinase R, viral polymerase, and Src homology 3 signaling proteins binding motif. Other hot loops were predicted in the V3 region and in the single-stranded DNA-binding protein motif. The different mechanisms by which the NS5A protein leads to immune system signaling dysfunction points to the natural genetic engineering of this protein.

Crystal structure of a trimeric form of the KV7.1 (KCNQ1) A‐domain tail coiled‐coil reveals structural plasticity and context dependent changes in a putative coiled‐coil trimerization motif

Coiled-coils are widespread protein-protein interaction motifs typified by the heptad repeat (abcdefg)(n) in which "a" and "d" positions are hydrophobic residues. Although identification of likely coiled-coil sequences is robust, prediction of strand order remains elusive. We present the X-ray crystal structure of a short form (residues 583-611), "Q1-short," of the coiled-coil assembly specificity domain from the voltage-gated potassium channel Kv7.1 (KCNQ1) determined at 1.7 A resolution. Q1-short lacks one and half heptads present in a previously studied tetrameric coiled-coil construct, Kv7.1 585-621, "Q1-long." Surprisingly, Q1-short crystallizes as a trimer. In solution, Q1-short self-assembles more poorly than Q1-long and depends on an R-h-x-x-h-E motif common to trimeric coiled-coils. Addition of native sequences that include "a" and "d" positions C-terminal to Q1-short overrides the R-h-x-x-h-E motif influence and changes assembly state from a weakly associated trimer to a strongly associated tetramer. These data provide a striking example of a naturally occurring amino sequence that exhibits context-dependent folding into different oligomerization states, a three-stranded versus a four-stranded coiled-coil. The results emphasize the degenerate nature of coiled-coil energy landscapes in which small changes can have drastic effects on oligomerization. Discovery of these properties in an ion channel assembly domain and prevalence of the R-h-x-x-h-E motif in coiled-coil assembly domains of a number of different channels that are thought to function as tetrameric assemblies raises the possibility that such sequence features may be important for facilitating the assembly of intermediates en route to the final native state.

BTB protein, dKLHL18/CG3571, serves as an adaptor subunit for a dCul3 ubiquitin ligase complex

The BTB domain is a highly conserved protein-protein interaction motif and functions in diverse cellular processes, including transcriptional regulation, ion channel assembly, cytoskeleton dynamics and apoptosis. Recently, it was reported that some BTB domain-containing proteins associate with Cullin-3 (Cul3), an E3 ubiquitin ligase, and act as an adaptor for Cul3 recognition of its substrate. However, the target substrates for the Cul3/BTB protein E3 ubiquitin ligase complex are largely unknown. Here, we report the characterization of a novel Drosophila BTB protein, dKLHL18/CG3571. By purification of a dKLHL18-associated complex, we identified CG10324, CG5808, l(2)37Cb and dCul3/guftagu. Indeed, the physical association of dKLHL18 with these proteins was observed in insect S2 cells, and genetic interactions among the identified factors were also observed in thorax development. Moreover, transient overexpression of dKLHL18 increased the ubiquitinated protein levels of CG10324 and CG5808. These findings suggest that dKLHL18 is an adaptor for a dCul3 E3 ubiquitin ligase to accommodate CG10324, CG5808 and l(2)37Cb proteins for ubiquitination.