Conjugation of the ubiquitin-like modifier Nedd8 to cullins is critical for the function of SCF-type ubiquitin ligases and thus facilitates ubiquitin conjugation and ultimately degradation of SCF substrates, including several cell cycle regulators. Like ubiquitin, Nedd8 is produced as a precursor that must first be processed before it becomes active. In Saccharomyces cerevisiae this is carried out exclusively by the enzyme Yuh1.
Here we show that in the fission yeast, Schizosaccharomyces pombe, the Yuh1 orthologue, Uch1, is not the sole Nedd8 processing enzyme. Instead it appears that deubiquitylating enzymes can efficiently process the Nedd8 precursor in vivo.
Several enzymes contribute to Nedd8 precursor processing including a number of deubiquitylating enzymes.
Ubiquitin; Nedd8; Rub1; Cullin; Protein degradation; Precursor processing
Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli.
Immobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 μM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1.
Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.
Heparin cofactor II; Thrombin; Serpins; Exosites; Coagulation; Inhibition
Receptor tyrosine kinases (RTKs) are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis.
We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2) in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence) served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution.
Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins) and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.
Tyrosine kinase; Choanoflagellate; Receptor; SH2 domain
There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear.
Presentation of the hypothesis
In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels.
Testing the hypothesis
Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding.
Implications of the hypothesis
The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined.
Hepatoma-derived growth factor (HDGF) is a protein which is highly expressed in a variety of tumours. HDGF has mitogenic, angiogenic, neurotrophic and antiapoptotic activity but the molecular mechanisms by which it exerts these activities are largely unknown nor has its biological function in tumours been elucidated. Mass spectrometry was performed to analyse the HDGFStrep-tag interactome. By Pull–down-experiments using different protein and nucleic acid constructs the interaction of HDGF and nucleolin was investigated further.
A number of HDGFStrep-tag copurifying proteins were identified which interact with RNA or are involved in the cellular DNA repair machinery. The most abundant protein, however, copurifying with HDGF in this approach was nucleolin. Therefore we focus on the characterization of the interaction of HDGF and nucleolin in this study. We show that expression of a cytosolic variant of HDGF causes a redistribution of nucleolin into the cytoplasm. Furthermore, formation of HDGF/nucleolin complexes depends on bcl-2 mRNA. Overexpression of full length bcl-2 mRNA increases the number of HDGF/nucleolin complexes whereas expression of only the bcl-2 coding sequence abolishes interaction completely. Further examination reveals that the coding sequence of bcl-2 mRNA together with either the 5′ or 3′ UTR is sufficient for formation of HDGF/nucleolin complexes. When bcl-2 coding sequence within the full length cDNA is replaced by a sequence coding for secretory alkaline phosphatase complex formation is not enhanced.
The results provide evidence for the existence of HDGF and nucleolin containing nucleoprotein complexes which formation depends on the presence of specific mRNAs. The nature of these RNAs and other components of the complexes should be investigated in future.
Nucleolin; Bcl-2; Ribonucleoproteins
The ubiquitin ligase COP1, COnstitutively Photomorphogenic 1, functions in many biological responses in mammalian cells, but its downstream pathway remains unclear.
Here, we identified FIP200, a key regulator of mammalian autophagy, as a novel COP1-interacting protein by yeast two-hybrid screening. The interaction was confirmed by a GST-pulldown assay. Split-GFP analysis revealed that interaction between COP1 and FIP200 predominantly occurred in the cytoplasm and was enhanced in cells treated with UV irradiation. Different forms of FIP200 protein were expressed in cultured mammalian cells, and ectopic expression of COP1 reduced one of such forms.
These data suggest that COP1 modulates FIP200-associated activities, which may contribute to a variety of cellular functions that COP1 is involved in.
COP1; FIP200(RB1CC1); UV; Autophagy
The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity.
We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3Kα, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3Kα with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3Kα. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3Kα affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3Kα.
Phosphorylation of p85α S608 is not a significant regulator of wild-type or oncogenic PI3Kα lipid kinase activity.
PI3K; PIK3CA; Phosphoinositide; Kinase; Mutation; Oncogene; Phosphorylation
An important controversy in the relationship between beef tenderness and muscle characteristics including biochemical traits exists among meat researchers. The aim of this study is to explain variability in meat tenderness using muscle characteristics and biochemical traits available in the Integrated and Functional Biology of Beef (BIF-Beef) database. The BIF-Beef data warehouse contains characteristic measurements from animal, muscle, carcass, and meat quality derived from numerous experiments. We created three classes for tenderness (high, medium, and low) based on trained taste panel tenderness scores of all meat samples consumed (4,366 observations from 40 different experiments). For each tenderness class, the corresponding means for the mechanical characteristics, muscle fibre type, collagen content, and biochemical traits which may influence tenderness of the muscles were calculated.
Our results indicated that lower shear force values were associated with more tender meat. In addition, muscles in the highest tenderness cluster had the lowest total and insoluble collagen contents, the highest mitochondrial enzyme activity (isocitrate dehydrogenase), the highest proportion of slow oxidative muscle fibres, the lowest proportion of fast-glycolytic muscle fibres, and the lowest average muscle fibre cross-sectional area. Results were confirmed by correlation analyses, and differences between muscle types in terms of biochemical characteristics and tenderness score were evidenced by Principal Component Analysis (PCA). When the cluster analysis was repeated using only muscle samples from m. Longissimus thoracis (LT), the results were similar; only contrasting previous results by maintaining a relatively constant fibre-type composition between all three tenderness classes.
Our results show that increased meat tenderness is related to lower shear forces, lower insoluble collagen and total collagen content, lower cross-sectional area of fibres, and an overall fibre type composition displaying more oxidative fibres than glycolytic fibres.
Tenderness; Beef; Meta-analysis; Muscle biochemistry
Presenilin-1 (PS1) is a transmembrane protein first discovered because of its association with familial Alzheimer’s disease. Mice with null mutations in PS1 die shortly after birth exhibiting multiple CNS and non-CNS abnormalities. One of the most prominent features in the brains of PS1−/− embryos is a vascular dysgenesis that leads to multiple intracerebral hemorrhages. The molecular and cellular basis for the vascular dysgenesis in PS1−/− mice remains incompletely understood. Because the extracellular matrix plays key roles in vascular development we hypothesized that an abnormal extracellular matrix might be present in endothelial cells lacking PS1 and examined whether the lack of PS1 affects expression of fibronectin a component of the extracellular matrix known to be essential for vascular development.
We report that primary as well as continuously passaged PS1−/− endothelial cells contain more fibronectin than wild type cells and that the excess fibronectin in PS1−/− endothelial cells is incorporated into a fibrillar network. Supporting the in vivo relevance of this observation fibronectin expression was increased in microvascular preparations isolated from E14.5 to E18.5 PS1−/− embryonic brain. Reintroduction of PS1 into PS1−/− endothelial cells led to a progressive decrease in fibronectin levels showing that the increased fibronectin in PS1−/− endothelial cells was due to loss of PS1. Increases in fibronectin protein in PS1−/− endothelial cells could not be explained by increased levels of fibronectin RNA nor based on metabolic labeling studies by increased protein synthesis. Rather we show based on the rate of turnover of exogenously added biotinylated fibronectin that increased fibronectin in PS1−/− endothelial cells results from a slower degradation of the fibronectin fibrillar matrix on the cell surface.
These studies show that PS1 regulates the constitutive turnover of the fibronectin matrix in endothelial cells. These studies provide molecular clues that may help to explain the origin of the vascular dysgenesis that develops in PS1−/− embryonic mice.
Endothelial cells; Extracellular matrix; Fibronectin; Presenilin-1; Vascular development
Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases — including CaMKI, CaMKIV, and AMPK— to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKIα (Thr177) and of AMPK (Thr172) in vitro. Kinetic analysis indicated that the Km values of CaMKK isoforms for GTP (400-500 μM) were significantly higher than those for ATP (~15 μM), and a 2- to 4-fold decrease in Vmax was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of ~45 kDa and ~35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.
Calmodulin; CaMKK; Phosphate donor; GTP; Phosphorylation
Plant sucrose uptake transporters (SUTs) are H+/sucrose symporters related to the major facilitator superfamily (MFS). SUTs are essential for plant growth but little is known about their transport mechanism. Recent work identified several conserved, charged amino acids within transmembrane spans (TMS) in SUTs that are essential for transport activity. Here we further evaluated the role of one of these positions, R188 in the fourth TMS of OsSUT1, a type II SUT.
The OsSUT1(R188K) mutant, studied by expression in plants, yeast, and Xenopus oocytes, did not transport sucrose but showed a H+ leak that was blocked by sucrose. The H+ leak was also blocked by β-phenyl glucoside which is not translocated by OsSUT1. Replacing the corresponding Arg in type I and type III SUTs, AtSUC1(R163K) and LjSUT4(R169K), respectively, also resulted in loss of sucrose transport activity. Fluorination at the glucosyl 3 and 4 positions of α-phenyl glucoside greatly decreased transport by wild type OsSUT1 but did not affect the ability to block H+ leak in the R188K mutant.
OsSUT1 R188 appears to be essential for sucrose translocation but not for substrate interaction that blocks H+ leak. Therefore, we propose that an additional binding site functions in the initial recognition of substrates. The corresponding Arg in type I and III SUTs are equally important. We propose that R188 interacts with glucosyl 3-OH and 4-OH during translocation.
Sucrose transporter; Major facilitator superfamily; Substrate binding; Mutagenesis
Nasopharyngeal carcinoma (NPC) has a high metastatic feature. N,N′-Dinitrosopiperazine (DNP) is involved in NPC metastasis, but its mechanism is not clear. The aim of this study is to reveal the pathogenesis of DNP-involved metastasis. 6-10B cells with low metastasis are from NPC cell line SUNE-1, were used to investigate the mechanism of DNP-mediated NPC metastasis.
6-10B cells were grown in DMEM containing 2H4-L-lysine and 13C 6 15 N4-L-arginine or conventional L-lysine and L-arginine, and identified the incorporation of amino acid by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Labeled 6-10B cells were treated with DNP at 0 -18 μM to establish the non-cytotoxic concentration (NCC) range. NCC was 0 -10 μM. Following treatment with DNP at this range, the motility and invasion of cells were detected in vitro, and DNP-mediated metastasis was confirmed in the nude mice. DNP increased 6-10B cell metastasis in vitro and vivo. DNP-induced protein expression was investigated using a quantitative proteomic. The SILAC-based approach quantified 2698 proteins, 371 of which showed significant change after DNP treatment (172 up-regulated and 199 down-regulated proteins). DNP induced the change in abundance of mitochondrial proteins, mediated the status of oxidative stress and the imbalance of redox state, increased cytoskeletal protein, cathepsin, anterior gradient-2, and clusterin expression. DNP also increased the expression of secretory AKR1B10, cathepsin B and clusterin 6-10B cells. Gene Ontology and Ingenuity Pathway analysis showed that DNP may regulate protein synthesis, cellular movement, lipid metabolism, molecular transport, cellular growth and proliferation signaling pathways.
DNP may regulate cytoskeletal protein, cathepsin, anterior gradient-2, and clusterin expression, increase NPC cells motility and invasion, is involved NPC metastasis.
Dinitrosopiperazine; Carcinogen; Nasopharyngeal carcinoma; Metastasis; Quantitative proteomics
Standard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP) -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD) is still missing.
Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult.
We developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM) was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8) was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S)-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED) and was used to systematically analyze functionally and structurally relevant positions in the superfamily of ThDP-dependent decarboxylases.
The numbering scheme serves as a tool for the reliable sequence alignment of ThDP-dependent decarboxylases and the unambiguous identification and communication of corresponding positions. Thus, it is the basis for the systematic and automated analysis of sequence-encoded properties such as structural and functional relevance of amino acid positions, because the analysis of conserved positions, the identification of correlated mutations and the determination of subfamily specific amino acid distributions depend on reliable multisequence alignments and the unambiguous identification of the alignment columns. The method is reliable and robust and can easily be adapted to further protein families.
Carnitine Palmitoyltransferase-1c (CPT1c) is a neuron specific homologue of the carnitine acyltransferase family of enzymes. CPT1 isoenzymes transfer long chain acyl groups to carnitine. This constitutes a rate setting step for mitochondrial fatty acid beta-oxidation by facilitating the initial step in acyl transfer to the mitochondrial matrix. In general, neurons do not heavily utilize fatty acids for bioenergetic needs and definitive enzymatic activity has been unable to be demonstrated for CPT1c. Although there are studies suggesting an enzymatic role of CPT1c, its role in neurochemistry remains elusive.
In order to better understand how CPT1c functions in neural metabolism, we performed unbiased metabolomic profiling on wild-type (WT) and CPT1c knockout (KO) mouse brains. Consistent with the notion that CPT1c is not involved in fatty acid beta-oxidation, there were no changes in metabolites associated with fatty acid oxidation. Endocannabinoids were suppressed in the CPT1c KO, which may explain the suppression of food intake seen in CPT1c KO mice. Although products of beta-oxidation were unchanged, small changes in carnitine and carnitine metabolites were observed. Finally, we observed changes in redox homeostasis including a greater than 2-fold increase in oxidized glutathione. This indicates that CPT1c may play a role in neural oxidative metabolism.
Steady-state metabolomic analysis of CPT1c WT and KO mouse brains identified a small number of metabolites that differed between CPT1c WT and KO mice. The subtle changes in a broad range of metabolites in vivo indicate that CPT1c does not play a significant or required role in fatty acid oxidation; however, it could play an alternative role in neuronal oxidative metabolism.
Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative.
We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity.
Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation.
Methionine; Methionine sulfoxide; Cysteine; Methionine sulfoxide reductase; Protein oxidation and reduction; Protein repair; Antibodies
Phosphatase of regenerating liver-3 (PRL-3 or PTP4A3) has been implicated in controlling cancer cell proliferation, motility, metastasis, and angiogenesis. Deregulated expression of PRL-3 is highly correlated with cancer progression and predicts poor survival. Although PRL-3 was categorized as a tyrosine phosphatase, its cellular substrates remain largely unknown.
We demonstrated that PRL-3 interacts with integrin β1 in cancer cells. Recombinant PRL-3 associates with the intracellular domain of integrin β1 in vitro. Silencing of integrin α1 enhances PRL-3-integrin β1 interaction. Furthermore, PRL-3 diminishes tyrosine phosphorylation of integrin β1 in vitro and in vivo. With site-specific anti-phosphotyrosine antibodies against residues in the intracellular domain of integrin β1, tyrosine-783, but not tyrosine-795, is shown to be dephosphorylated by PRL-3 in a catalytic activity-dependant manner. Phosphorylation of Y783 is potentiated by ablation of PRL-3 or by treatment with a chemical inhibitor of PRL-3. Conversely, depletion of integrin α1 decreases the phosphorylation of this site.
Our results revealed a direct interaction between PRL-3 and integrin β1 and characterized Y783 of integrin β1 as a bona fide substrate of PRL-3, which is negatively regulated by integrin α1.
PRL-3; tyrosine phosphatase; integrin β1; dephosphorylation
STAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE) 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood.
Here, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1.
Our results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.
Signal transduction; Transcription factors; Sumoylation; Signal transducers and activators of transcription (STATs); Interferon
Streptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.
In this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.
We developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.
Sialidase; Neuraminidase; Chromogenic sialic acids; Kinetic assay; Streptococcus pneumoniae
Mutations to the TSC1 and TSC2 genes cause the disease tuberous sclerosis complex. The TSC1 and TSC2 gene products form a protein complex that integrates multiple metabolic signals to regulate the activity of the target of rapamycin (TOR) complex 1 (TORC1) and thereby control cell growth. Here we investigate the quaternary structure of the TSC1-TSC2 complex by gel filtration and coimmunoprecipitation.
TSC1 and TSC2 co-eluted in high molecular weight fractions by gel filtration. Coimmunoprecipitation of distinct tagged TSC1 and TSC2 isoforms demonstrated that TSC1-TSC2 complexes contain multiple TSC1 and TSC2 subunits.
TSC1 and TSC2 interact to form large complexes containing multiple TSC1 and TSC2 subunits.
TSC1; TSC2; TSC1-TSC2 complex; Quaternary structure
The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software.
The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R.
Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.
Ischemia repurfusion injury; Two dimensional gel electrophoresis; Mass spectrometry; Liver transplantation; Chaperonins; Endoplasmic reticulum (ER) stress
Protein-protein interactions are at the basis of many cellular processes, and they are also involved in the interaction between pathogens and their host(s). Many intracellular pathogenic bacteria translocate proteins called effectors into the cytoplasm of the infected host cell, and these effectors can interact with one or several host protein(s). An effector named RicA was recently reported in Brucella abortus to specifically interact with human Rab2 and to affect intracellular trafficking of this pathogen.
In order to identify regions of the RicA protein involved in the interaction with Rab2, RicA was subjected to extensive random mutagenesis using error prone polymerase chain reaction. The resulting allele library was selected by the yeast two-hybrid assay for Rab2-interacting clones that were isolated and sequenced, following the “absence of interference” approach. A tridimensional model of RicA structure was used to position the substitutions that did not affect RicA-Rab2 interaction, giving a “negative image” of the putative interaction region. Since RicA is a bacterial conserved protein, RicA homologs were also tested against Rab2 in a yeast two-hybrid assay, and the C. crescentus homolog of RicA was found to interact with human Rab2. Analysis of the RicA structural model suggested that regions involved in the folding of the “beta helix” or an exposed loop with the IGFP sequence could also be involved in the interaction with Rab2. Extensive mutagenesis of the IGFP loop suggested that loss of interaction with Rab2 was correlated with insolubility of the mutated RicA, showing that “absence of interference” approach also generates surfaces that could be necessary for folding.
Extensive analysis of substitutions in RicA unveiled two structural elements on the surface of RicA, the most exposed β-sheet and the IGFP loop, which could be involved in the interaction with Rab2 and protein folding. Our analysis of mutants in the IGFP loop suggests that, at least for some mono-domain proteins such as RicA, protein interaction analysis using allele libraries could be complicated by the dual effect of many substitutions affecting both folding and protein-protein interaction.
Protein-protein interaction; Yeast two-hybrid; Mutagenesis; Brucella
It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings.
We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP.
The data outlined serves to demonstrate the “push” mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved “push” and “pull” mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the γ-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.
Epitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology. The knowledge collected on the subdomain organization of the two subunits of the adhesion complex dystroglycan (DG) enabled us to insert the 10 amino acids myc-tag at different locations along the α-subunit, in order to better visualize and investigate the DG complex in eukaryotic cells.
We have generated two forms of DG polypeptides via the insertion of the myc-tag 1) within a flexible loop (between a.a. 170 and 171) that separates two autonomous subdomains, and 2) within the C-terminal domain in position 500. Their analysis showed that double-tagging (the β-subunit is linked to GFP) does not significantly interfere with the correct processing of the DG precursor (pre-DG) and confirmed that the α-DG N-terminal domain is processed in the cell before α-DG reaches its plasma membrane localization. In addition, myc insertion in position 500, right before the second Ig-like domain of α-DG, proved to be an efficient tool for the detection and pulling-down of glycosylated α-DG molecules targeted at the membrane.
Further characterization of these and other myc-permissive site(s) will represent a valid support for the study of the maturation process of pre-DG and could result in the creation of a new class of intrinsic doubly-fluorescent DG molecules that would allow the monitoring of the two DG subunits, or of pre-DG, in cells without the need of antibodies.
Thirteen serotypes of Shigella flexneri (S. flexneri) have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM) regions.
In this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines–R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52–53, GS 138–139 and SYG 274–276), phenylalanine-proline motifs (FP 78–79 and FPV 282–84) and a tryptophan-threonine motif (WT141-142) found in TM segments and residues RK 110–111, GR 269–270 and D333 found in hydrophilic loops were also found to be critical to Oac function.
By studying the effect of the mutations on Oac’s function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110–111, GR 269–270 and D333) were shown to have an affect on Oac assembly which suggests a structural role for these motifs. The phenylalanine-proline and the tryptophan-threonine motifs affect Oac function which could suggest a catalytic role for these amino acids.
Shigella flexneri; O-acetyltransferase (Oac); Critical amino acids
Thymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies.
Canine TK1 phosphorylated the thymidine (dThd) analog 3'-azido-thymidine (AZT) as efficiently as it did dThd, whereas AZT phosphorylation by human TK1 was less efficient than that of dThd. Dog TK1 was also more thermostable and pH tolerant than the human enzyme. Oligomeric forms were observed with both enzymes in addition to the tetrameric and dimeric forms. Cellular TK1 was predominantly seen in dimeric and tetrameric forms, in the case of both dog TK1 from MDCK cells and human TK1 from CEM cells. Active serum TK1 was found mainly in a high molecular weight form, and treatment with a reducing agent shifted the high molecular weight complex to lower molecular weight forms with reduced total activity. Western blot analysis demonstrated a polypeptide of 26 kDa (dog) and 25 kDa (human) for cellular and serum TK1. There was no direct correlation between serum TK1 activity and protein level. It appears that a substantial fraction of serum TK1 is not enzymatically active.
These results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. The results presented here provide important information for the future development and use of serum TK1 as a diagnostic biomarker in human and veterinary medicine.
Recombinant Thymidine Kinase 1; Canine; Human; AZT; Serum Thymidine Kinase 1