Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme

U8 snoRNP is required for accumulation of mature 5.8S and 28S rRNA in vertebrates. We are identifying proteins that bind U8 RNA with high specificity to understand how U8 functions in ribosome biogenesis. Here, we characterize a Xenopus 29 kDa protein (X29), which we previously showed binds U8 RNA with high affinity. X29 and putative homologs in other vertebrates contain a NUDIX domain found in MutT and other nucleotide diphosphatases. Recombinant X29 protein has diphosphatase activity that removes m(7)G and m(227)G caps from U8 and other RNAs in vitro; the putative 29 kDa human homolog also displays this decapping activity. X29 is primarily nucleolar in Xenopus tissue culture cells. We propose that X29 is a member of a conserved family of nuclear decapping proteins that function in regulating the level of U8 snoRNA and other nuclear RNAs with methylated caps.     

Identification of a novel hepatic calcium binding protein

Calcium binding activity in the 100,000 X g supernatant of bovine liver has been isolated by a procedure involving DEAE cellulose and Sephadex G-100 chromatography. In addition to calmodulin, two new high affinity calcium binding proteins have been identified. On gel filtration chromatography these proteins migrate with apparent molecular weights of 83,700 and 51,400; whereas by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the two proteins migrate identically with Mr 63,000. In the presence of millimolar Mg2+, both proteins bind up to one mol Ca2+/mol protein. Half-maximal binding occurs at approximately 0.1 microM Ca2+. Amino acid compositional analysis reveals that both proteins are acidic, and contain about 40% glx and asx. Peptide mapping procedures suggest that these proteins may be highly homologous or multiple forms of a single protein. The results show the existence of calcium binding protein(s) other than calmodulin in hepatic cytosol.     

Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA.

The interaction between the U1 snRNP-specific U1 A protein and U1 snRNA has been analysed. The binding site for the protein on the RNA is shown to be in hairpin II, which extends from positions 48 to 91 in the RNA. Within this hairpin the evolutionarily conserved loop sequence is crucial for interaction with U1 A protein. U1 A protein can also bind the loop sequence when it is part of an artificial RNA which cannot form a stable hairpin structure. The region of the protein required to bind to U1 snRNA consists of a conserved 80 amino acid motif, previously identified in many ribonucleoprotein (RNP) proteins, together with (maximally) 11 N-terminal and 10 C-terminal flanking amino acids. Point mutations introduced into two of the most highly conserved regions of this motif abolish RNA binding. U1 snRNA mutants from which the U1 A binding site has been deleted are shown to be capable of assembly into RNP particles which are immunoprecipitable by patient antisera which recognize U1 A protein. The role of RNA-protein and protein-protein interactions in U snRNP assembly are discussed.     

Identification of a novel Mcl-1 protein binding motif

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.     

Identification of a carcinoembryonic antigen binding protein on monocytes

Tumor-associated monocyte/macrophage cells are important stromal components involved in tumor development. A protein on human monocyte is identified that binds to carcinoembryonic antigen (CEA), a glycoprotein overexpressed in colon tumors. This implicates a role for this protein in CEA processing and establishes a link between monocytes and colon tumor cells. In vitro uptake of 125I-labeled CEA with isolated monocytes showed time and temperature dependence. The binding of 125I-CEA was specific and saturable as it could be inhibited by an excess of unlabeled CEA. To identify the binding protein on monocyte, we used a radiolabeled photoactivable heterobifunctional crosslinking agent and demonstrated that CEA reacts with a 115kDa protein as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. Treatment of human monocytes in vitro with CEA resulted in a several fold increase in the production of proinflammatory cytokines TNF-alpha, IL-1 beta, and IL-6 compared to untreated controls. Binding of CEA to the monocyte protein may have implications in colon tumorigenesis.     

Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein

The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.     

Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

Identification of a novel calcium binding protein from bovine brain

A novel Ca²⁺ binding protein, named caligulin, was extracted from the heat-treated 100 000 × g supernatant of bovine brain and purified to electrophoretic homogeneity. The apparent M_r of caligulin determined on sodium dodecyl sulfate polyacrylamide gels was 24 000. Analysis by gel filtration chromatography indicated an apparent M_r of 33 000, suggesting a monomeric protein. Amino acid composition data demonstrated the presence of 25% acidic residues, 12% basic residues and 10% leucine. In the presence of 1 mM MgCl₂ and 0.15 M KCl, caligulin bound 1 mol Ca²⁺/mol protein with half-maximal binding at about 0.2 μM Ca²⁺.     

Identification of a co-repressor binding site in catabolite control protein CcpA

The catabolite control protein CcpA is the central regulator of carbon catabolite repression in Bacilli and other Gram-positive bacteria. A comparison of 12 CcpA-like sequences with regulators from the LacI/GalR family defines a CcpA subfamily based on extensive similarities found among CcpAs and not in 32 other members of the family. These amino acids are clustered in three blocks in the CcpA sequence. Their interpretation, assuming a PurR-like fold, reveals that almost all of them are surface exposed and form a continuous patch on the N-terminal subdomain of the protein core extending into the DNA reading head. We introduced nine single amino acid exchanges in the subfamily specific residues of CcpA from Bacillus megaterium . Six mutants, namely CcpA47RS, 79AE, 89YE, 295YR, 299YE and 303RD, are inactive or severely impaired in catabolite repression, underlining their relevance for CcpA function. They are negatively transdominant over wild-type CcpA demonstrating their ability to correctly fold for dimerization. Five of them are unable or impaired in binding HPr-Ser-46-P in vitro , establishing a correlation between catabolite repression efficiency and HPr-Ser-46-P binding. These results support the hypothesis that the conserved region in CcpA is the HPr-Ser-46-P binding site.     

Identification of calnexin as a binding protein for Amadori-modified glycated albumin

Albumin modified by Amadori glucose adducts (glycated albumin) selectively binds to glomerular mesangial cells and triggers signal transduction processes that modulate cellular function. To identify glycated albumin binding proteins, we applied membrane extracts prepared from murine mesangial cells to a column of lysine-Sepharose followed by application to an affinity column of fructosyllysine-Sepharose. This procedure yielded an approximately 90 kDa polypeptide that immunoreacted with Amadori-modified but not carbohydrate-free albumin. MALDI mass fingerprinting matched 9 out of 25 peptides with calnexin, and amino acid analysis showed homology with this transmembrane calcium-binding protein of the calreticulin family. These results indicate that one of the mesangial cell receptors for glycated albumin is a calnexin-like protein.     

Identification of a high density lipoprotein binding protein from adrenocortical membranes

A protein band with an apparent molecular weight of 78,000 daltons has been identified in the solubilised plasma membrane extract of sheep adrenal cortex which binds HDL3 devoid of E apolipoprotein. Following 'Western' blotting, and development of the nitrocellulose strips with appropriate antisera and color reagent, the same band, unlike other cortical membrane proteins or albumin, bound AI and AII apolipoproteins. Human LDL bound weakly to the same band but more strongly to another two proteins of higher molecular weight. These studies confirm the same degree of specificity of HDL3 binding found with cultured adrenal cells and strengthen the suggested existence of a specific HDL receptor.     

Identification of a lipid A binding site in the acute phase reactant lipopolysaccharide binding protein

Lipopolysaccharide (LPS) binding protein (LBP), a recently discovered 60-kDa acute phase protein, is present in the acute phase serum of many species including human, rabbits, mice, and rats. Using either highly purified LBP from acute phase rabbit serum or unfractionated acute phase rabbit serum as a source of LBP, we examined the binding of LBP to LPS immobilized on plastic microtiter plates and to LPS electrotransferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of LBP bound to LPS was detected with goat anti-rabbit LBP and peroxidase-conjugated rabbit anti-goat IgG. LBP was found to bind to a variety of LPS types from both rough and smooth strains of Gram-negative bacteria, to lipid A, and to the tetraacyl glucosamine disaccharide diphosphate precursor IVA, but bound very poorly to the diacyl glucosamine phosphate, lipid X. No binding to 3-deoxyoctulosonic acid was observed. Binding affinities for LPS are near 10(9) M-1. The data presented here support the concept that LBP contains a binding site for lipid A.     

The U5 snRNA internal loop 1 is a platform for Brr2, Snu114 and Prp8 protein binding during U5 snRNP assembly

The U5 small nuclear ribonucleoprotein particle (snRNP) forms the heart of the spliceosome which is required for intron removal from pre‐mRNA. The proteins Prp8, Snu114 and Brr2 all assemble with the U5 small nuclear RNA (snRNA) to produce the U5 snRNP. Successful assembly of the U5 snRNP, then incorporation of this snRNP into the U4/U6.U5 tri‐snRNP and the spliceosome, is essential for producing an active spliceosome. We have investigated the requirements for Prp8, Snu114 and Brr2 association with the U5 snRNA to form the U5 snRNP in yeast. Mutations were constructed in the highly conserved loop 1 and internal loop 1 (IL1) of the U5 snRNA and their function assessed in vivo. The influence of these U5 mutations on association of Prp8, Snu114 and Brr2 with the U5 snRNA were then determined. U5 snRNA loop 1 and both sides of IL1 in U5 were important for association of Prp8, Snu114 and Brr2 with the U5 snRNA. Mutations in the 3′ side of U5 IL1 resulted in the greatest reduction of Prp8, Snu114 and Brr2 association with the U5 snRNA. Genetic screening of brr2 and U5 snRNA mutants revealed synthetic lethal interactions between alleles in Brr2 and the 3′ side of U5 snRNA IL1 which reflects reduced association between Brr2 and U5 IL1. We propose that the U5 snRNA IL1 is a platform for protein binding and is required for Prp8, Brr2 and Snu114 association with the U5 snRNA to form the U5 snRNP. J. Cell. Biochem. 114: 2770–2784, 2013. © 2013 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Identification of a novel inhibitory actin-capping protein binding motif in CD2-associated protein

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton.     

Identification of ATP binding residues of a protein from its primary sequence

Background  

One of the major challenges in post-genomic era is to provide functional annotations for large number of proteins arising from genome sequencing projects. The function of many proteins depends on their interaction with small molecules or ligands. ATP is one such important ligand that plays critical role as a coenzyme in the functionality of many proteins. There is a need to develop method for identifying ATP interacting residues in a ATP binding proteins (ABPs), in order to understand mechanism of protein-ligands interaction.