Tubby and tubby-like protein 1 are new MerTK ligands for phagocytosis

Tubby and tubby-like protein 1 (Tulp1) are newly identified phagocytosis ligands to facilitate retinal pigment epithelium (RPE) and macrophage phagocytosis. Both proteins without classical signal peptide have been demonstrated with unconventional secretion. Here, we characterized them as novel MerTK ligands to facilitate phagocytosis. Tulp1 interacts with Tyro3, Axl and MerTK of the TAM receptor tyrosine kinase subfamily, whereas tubby binds only to MerTK. Excessive soluble MerTK extracellular domain blocked tubby- or Tulp1-mediated phagocytosis. Both ligands induced MerTK activation with receptor phosphorylation and signalling cascade, including non-muscle myosin II redistribution and co-localization with phagosomes. Tubby and Tulp1 are bridging molecules with their N-terminal region as MerTK-binding domain and C-terminal region as phagocytosis prey-binding domain (PPBD). Five minimal phagocytic determinants (MPDs) of K/R(X)(1-2)KKK in Tulp1 N-terminus were defined as essential motifs for MerTK binding, receptor phosphorylation and phagocytosis. PPBD was mapped to the highly conserved 54 amino acids at the C-terminal end of tubby and Tulp1. These data suggest that tubby and Tulp1 are novel bridging molecules to facilitate phagocytosis through MerTK.     

Efficient identification of tubby‐binding proteins by an improved system of T7 phage display

Mutation in the tubby gene causes adult‐onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby‐like protein 1 (Tulp1), whose C‐terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N‐terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N‐terminus (tubby‐N) as bait to identify unknown binding proteins with open‐reading‐frame (ORF) phage display. T7 phage display was engineered with three improvements: high‐quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait‐binding proteins in as fast as ～4–7 days. While phage display with conventional cDNA libraries identifies high percentage of out‐of‐frame unnatural short peptides, all 28 tubby‐N‐binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two‐hybrid assay and protein pull‐down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby‐specific binding protein. These data suggest that tubby‐N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly‐engineered ORF phage display is a powerful technology to identify unknown protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of tubby and tubby-like protein 1 as eat-me signals by phage display

Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1^−/− mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.     

Differential Roles of Tubby Family Proteins in Ciliary Formation and Trafficking

Cilia are highly specialized organelles that extend from the cell membrane and function as cellular signaling hubs. Thus, cilia formation and the trafficking of signaling molecules into cilia are essential cellular processes. TULP3 and Tubby (TUB) are members of the tubby-like protein (TULP) family that regulate the ciliary trafficking of G-protein coupled receptors, but the functions of the remaining TULPs (i.e., TULP1 and TULP2) remain unclear. Herein, we explore whether these four structurally similar TULPs share a molecular function in ciliary protein trafficking. We found that TULP3 and TUB, but not TULP1 or TULP2, can rescue the defective cilia formation observed in TULP3-knockout (KO) hTERT RPE-1 cells. TULP3 and TUB also fully rescue the defective ciliary localization of ARL13B, INPP5E, and GPR161 in TULP3 KO RPE-1 cells, while TULP1 and TULP2 only mediate partial rescues. Furthermore, loss of TULP3 results in abnormal IFT140 localization, which can be fully rescued by TUB and partially rescued by TULP1 and TULP2. TUB’s capacity for binding IFT-A is essential for its role in cilia formation and ciliary protein trafficking in RPE-1 cells, whereas its capacity for PIP₂ binding is required for proper cilia length and IFT140 localization. Finally, chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescues ciliary protein trafficking, but not cilia formation. Together, these two TULP domains play distinct roles in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play other unknown molecular roles that should be addressed in the future.     

Tubby proteins: the plot thickens

The tubby mouse, which shows late-onset obesity and neurosensory deficits, arises from a mutation in the Tub gene. Tub shares homology with the genes for tubby-like proteins Tulp1, Tulp2 and Tulp3. Ablation of Tub, Tulp1 or Tulp3 causes disease phenotypes that are indicative of their importance in nervous-system function and development. Despite this importance, the biochemical functions of tubby-like proteins are only now beginning to be understood. At present, data indicate that tubby-like proteins might function as heterotrimeric-G-protein-responsive intracellular signalling factors, although an array of data also implicates them in other processes.     

The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein alpha-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal beta-TM). The interaction between Enigma and skeletal beta-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal beta-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal beta-TM in transfected cells. The association of Enigma with skeletal beta-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells.     

Regulation of progenitor cell survival by a novel chromatin remodeling factor during neural tube development

During development, progenitor cell survival is essential for proper tissue functions, but the underlying mechanisms are not fully understood. Here we show that ERCC6L2, a member of the Snf2 family of helicase-like proteins, plays an essential role in the survival of developing chick neural cells. ERCC6L2 expression is induced by the Sonic Hedgehog (Shh) signaling molecule by a mechanism similar to that of the known Shh target genes Ptch1 and Gli1. ERCC6L2 blocks programmed cell death induced by Shh inhibition and this inhibition is independent of neural tube patterning. ERCC6L2 knockdown by siRNA resulted in the aberrant appearance of apoptotic cells. Furthermore, ERCC6L2 cooperates with the Shh signal and plays an essential role in the induction of the anti-apoptotic factor Bcl-2. Taken together, ERCC6L2 acts as a key factor in ensuring the survival of neural progenitor cells.     

Molecular analyses of the rice tubby-like protein gene family and their response to bacterial infection

Tubby-like protein family has been identified in various multicellular organisms, indicating its fundamental functions in the organisms. However, the roles of plant tubby-like proteins are unknown. In this study, we have defined the tubby-like protein gene (OsTLP) family with 14 members in rice. Most of the OsTLPs harbor a tubby domain in their carboxyl terminus and an F-box domain in the amino terminus. The expression of all the OsTLPs was induced on infection of Xanthomonas oryzae pv. oryzae, which causes bacterial blight, one of the most devastating diseases of rice worldwide. The maximal expression levels were observed at 2–8 h after infection for all the genes. Eight of the 14 OsTLPs were also responsive to wounding. All the OsTLPs showed differential expression in different tissues at different developmental stages. However, four pairs of the 14 OsTLPs, with each pair having high sequence similarity and distributing on the similar position of different chromosomes, showed similar expression pattern in different tissues, indicating their direct relationship in evolution. These results suggest that the OsTLP family is involved in host–pathogen interaction and it may be also associated with other physiological and developmental activities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Unconventional secretion of tubby and tubby-like protein 1

Tubby-like proteins (Tulps) with no signal peptide have been characterized as cytoplasmic proteins with various intracellular functions, including binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. PI(4,5)P₂ has been implicated in unconventional secretion of fibroblast growth factor-2 without a signal peptide. Here, we show that all Tulps are expressed intracellularly and extracellularly. Tubby secretion is partially dependent on its PI(4,5)P₂-binding activity with an essential secretory signal in the N-terminus. Pathogenic mutation in Tubby mice has no impact on tubby extracellular trafficking. Moreover, unconventional secretion of tubby and Tulp1 is independent of endoplasmic reticulum-Golgi pathway. These data implicate that Tulps may function extracellularly as well.     

Functional,structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum‐activated malate/anion transporter,TaALMT1

Triticum aestivum aluminum‐activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub‐group of root‐localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure–function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re‐examine the role of protein domains in terms of their potential involvement in the Al‐dependent enhancement (i.e. Al‐responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N‐domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C‐domain. However, segments in both domains are involved in Al³⁺ sensing. We identified two regions, one at the N‐terminus and a hydrophobic region at the C‐terminus, that jointly contribute to the Al‐response phenotype. Interestingly, the characteristic motif at the N‐terminus appears to be specific for Al‐responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure–function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al³⁺ sensing.     

Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways

ABSTRACT

Armadillo (ARM) repeat proteins constitute a large protein family with diverse and fundamental functions in all organisms, and armadillo repeat domains share high structural similarity. However, exactly how these structurally similar proteins can mediate diverse functions remains a long-standing question. Vac8 (vacuole related 8) is a multifunctional protein that plays pivotal roles in various autophagic pathways, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways in the budding yeast Saccharomyces cerevisiae. Vac8 comprises an H1 helix at the N terminus, followed by 12 armadillo repeats. Herein, we report the crystal structure of Vac8 bound to Atg13, a key component of autophagic machinery. The 70-Å extended loop of Atg13 binds to the ARM domain of Vac8 in an antiparallel manner. Structural, biochemical, and in vivo experiments demonstrated that the H1 helix of Vac8 intramolecularly associates with the first ARM and regulates its self-association, which is crucial for Cvt and PMN pathways. The structure of H1 helix-deleted Vac8 complexed with Atg13 reveals that Vac8[Δ19–33]-Atg13 forms a heterotetramer and adopts an extended superhelical structure exclusively employed in the Cvt pathway. Most importantly, comparison of Vac8-Nvj1 and Vac8-Atg13 provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related but distinct cellular pathways.     

Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog.

Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes. Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer. Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium. Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions. The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding. The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody. In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro. Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants. The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins.     

Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog

Mammalian glycerol uptake/transporter 1 (Gup1), a homolog of Saccharomyces cerevisiae Gup1, is predicted to be a member of the membrane-bound O-acyltransferase family and is highly homologous to mammalian hedgehog acyltransferase, known as Skn, the homolog of the Drosophila skinny hedgehog gene product. Although mammalian Gup1 has a sequence conserved among the membrane-bound O-acyltransferase family, the histidine residue in the motif that is indispensable to the acyltransferase activity of the family has been replaced with leucine. In this study, we cloned Gup1 cDNA from adult mouse lung and examined whether Gup1 is involved in the regulation of N-terminal palmitoylation of Sonic hedgehog (Shh). Subcellular localization of mouse Gup1 was indistinguishable from that of mouse Skn detected using the fluorescence of enhanced green fluorescent protein that was fused to each C terminus of these proteins. Gup1 and Skn were co-localized with an endoplasmic reticulum marker, 78 kDa glucose-regulated protein, suggesting that these two molecules interact with overlapped targets, including Shh. In fact, full-length Shh coprecipitated with FLAG-tagged Gup1 by immunoprecipitation using anti-FLAG IgG. Ectopic expression of Gup1 with full-length Shh in cells lacking endogenous Skn showed no hedgehog acyltransferase activity as determined using the monoclonal antibody 5E1, which was found to recognize the palmitoylated N-terminal signaling domain of Shh under denaturing conditions. On the other hand, Gup1 interfered with the palmitoylation of Shh catalyzed by endogenous Skn in COS7 and NSC34. These results suggest that Gup1 is a negative regulator of N-terminal palmitoylation of Shh and may contribute to the variety of biological actions of Shh.     

ZC3H12A/MCPIP1/Regnase‐1‐related endonucleases: An evolutionary perspective on molecular mechanisms and biological functions

The mammalian Zc3h12a/MCPIP1/Regnase‐1, an extensively studied regulator of inflammatory response, is the founding member of a ribonuclease family, which includes proteins related by the presence of the so‐called Zc3h12a‐like NYN domain. Recently, several related proteins have been described in Caenorhabditis elegans, allowing comparative evaluation of molecular functions and biological roles of these ribonucleases. We discuss the structural features of these proteins, which endow some members with ribonuclease (RNase) activity while others with auxiliary or RNA‐independent functions. We also consider their RNA specificity and highlight a common role for these proteins in cellular defense, which is remarkable considering the evolutionary distance and fundamental differences in cellular defense mechanisms between mammals and nematodes.     

Densin-180 interacts with delta-catenin/neural plakophilin-related armadillo repeat protein at synapses.

Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP (leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains) family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, we screened a yeast two-hybrid library using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and we isolated delta-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) as a Densin-180-interacting protein. delta-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimer's disease. Densin-180 PDZ domain binds the COOH terminus of delta-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with delta-catenin/NPRAP and N-cadherin. Although Densin-180 was reported to be a transmembrane protein, Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localized with delta-catenin/NPRAP at synapses in delta-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the delta-catenin/NPRAP-N-cadherin complex.     

The maize ID1 flowering time regulator is a zinc finger protein with novel DNA binding properties

The INDETERMINATE protein, ID1, plays a key role in regulating the transition to flowering in maize. ID1 is the founding member of a plant-specific zinc finger protein family that is defined by a highly conserved amino sequence called the ID domain. The ID domain includes a cluster of three different types of zinc fingers separated from a fourth C2H2 finger by a long spacer; ID1 is distinct from other ID domain proteins by having a much longer spacer. In vitro DNA selection and amplification binding assays and DNA binding experiments showed that ID1 binds selectively to an 11 bp consensus motif via the ID domain. Unexpectedly, site-directed mutagenesis of the ID1 protein showed that zinc fingers located at each end of the ID domain are not required for binding to the consensus motif despite the fact that one of these zinc fingers is a canonical C2H2 DNA binding domain. In addition, an ID1 in vitro deletion mutant that lacks the extra spacer between zinc fingers binds the same 11 bp motif as normal ID1, suggesting that all ID domain-containing proteins recognize the same DNA target sequence. Our results demonstrate that maize ID1 and ID domain proteins have novel zinc finger configurations with unique DNA binding properties.