Ser Mutation Research Articles

NfuA, a New Factor Required for Maturing Fe/S Proteins in Escherichia coli under Oxidative Stress and Iron Starvation Conditions

Iron/sulfur (Fe/S) proteins are central to the functioning of cells in both prokaryotes and eukaryotes. Here, we show that the yhgI gene, which we renamed nfuA, encodes a two-domain protein that is required for Fe/S biogenesis in Escherichia coli. The N-terminal domain resembles the so-called Fe/S A-type scaffold but, curiously, has lost the functionally important Cys residues. The C-terminal domain shares sequence identity with Nfu proteins. Mössbauer and UV-visible spectroscopic analyses revealed that, upon reconstitution, NfuA binds a [4Fe-4S] cluster. Moreover, NfuA can transfer this cluster to apo-aconitase. Mutagenesis studies indicated that the N- and C-terminal domains are important for NfuA function in vivo. Similarly, the functional importance of Cys residues present in the Nfu-like domain was demonstrated in vivo by introducing Cys-->Ser mutations. In vivo investigations revealed that the nfuA gene is important for E. coli to sustain oxidative stress and iron starvation. Also, combining nfuA with either isc or suf mutations led to additive phenotypic deficiencies, indicating that NfuA is a bona fide new player in Isc- and Suf-dependent Fe/S biogenesis pathways. Taken together, these data demonstrate that NfuA intervenes in the maturation of apoproteins in E. coli, allowing them to acquire Fe/S clusters. By taking into account results from numerous previous transcriptomic studies that had suggested a link between NfuA and protein misfolding, we discuss the possibility that NfuA could act as a scaffold/chaperone for damaged Fe/S proteins.

Modification of a human monoclonal antibody Fab fragment specific for Plasmodium falciparum 19-kDa C-terminal merozoite surface protein 1 by site-directed mutagenesis

We recently produced human monoclonal antibody Fab fragments specific for the 19-kDa C-terminal merozoite surface protein 1 of Plasmodium falciparum in a bacterial expression system. The effect of single amino acid modifications in the third complementarity-determining regions of the heavy and light chains on affinity was examined in one of the Fab fragments, Pf25. Recombination polymerase chain reaction was used to modify Tyr(92) or Ile(97) in the light chain and Val(101) or Trp(107) in the heavy chain. No effective replacements for Tyr(92) and Val(101) were found, but possible substitutions of Ile(97) with Gly, Leu, Glu, Ala and Ser, and of Trp(107) with Arg and Ser were demonstrated. Of these modified Fab fragments, the affinities of Fabs with Ile(97)-Leu and Trp(107)-Ser mutations were slightly higher than that of the original Fab. The effects of these modifications on the antigen-antibody interaction are discussed.

Mutagenesis of the crystal contact of acidic fibroblast growth factor.

An attempt has been made to improve a crystal contact of human acidic fibroblast growth factor (haFGF; 140 amino acids) to control the crystal growth, because haFGF crystallizes only as a thin-plate form, yielding crystals suitable for X-ray but not neutron diffraction. X-ray crystal analysis of haFGF showed that the Glu81 side chain, located at a crystal contact between haFGF molecules, is in close proximity with an identical residue related by crystallographic symmetry, suggesting that charge repulsion may disrupt suitable crystal-packing interactions. To investigate whether the Glu residue affects the crystal-packing interactions, haFGF mutants in which Glu81 was replaced by Ala, Val, Leu, Ser and Thr were constructed. Although crystals of the Ala and Leu mutants were grown as a thin-plate form by the same precipitant (formate) as the wild type, crystals of the Ser and Thr mutants were grown with increased thickness, yielding a larger overall crystal volume. X-ray structural analysis of the Ser mutant determined at 1.35 A resolution revealed that the hydroxy groups of Ser are linked by hydrogen bonds mediated by the formate used as a precipitant. This approach to engineering crystal contacts may contribute to the development of large protein crystals for neutron crystallography.

Predicting the Clinical Lethality of Osteogenesis Imperfecta from Collagen Glycine Mutations

Osteogenesis imperfecta (OI), or brittle bone disease, often results from missense mutation of one of the conserved glycine residues present in the repeating Gly-X-Y sequence characterizing the triple-helical region of type I collagen. A composite model was developed for predicting the clinical lethality resulting from glycine mutations in the alpha1 chain of type I collagen. The lethality of mutations in which bulky amino acids are substituted for glycine is predicted by their position relative to the N-terminal end of the triple helix. The effect of a Gly --> Ser mutation is modeled by the relative thermostability of the Gly-X-Y triplet on the carboxy side of the triplet containing the substitution. This model also predicts the lethality of Gly --> Ser and Gly --> Cys mutations in the alpha2 chain of type I collagen. The model was validated with an independent test set of six novel Gly --> Ser mutations. The hypothesis derived from the model of an asymmetric interaction between a Gly --> Ser mutation and its neighboring residues was tested experimentally using collagen-like peptides. Consistent with the prediction, a significant decrease in stability, calorimetric enthalpy, and folding time was observed for a peptide with a low-stability triplet C-terminal to the mutation compared to a similar peptide with the low-stability triplet on the N-terminal side. The computational and experimental results together relate the position-specific effects of Gly --> Ser mutations to the local structural stability of collagen and lend insight into the etiology of OI.

Structural Characterization of PTX3 Disulfide Bond Network and Its Multimeric Status in Cumulus Matrix Organization

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

Analyse moléculaire de la mutation p.Asn 370 Ser dans la maladie de Gaucher

Gaucher disease is one of the most prevalent lysosomal disorders. In this present study, we report a diagnostic strategy of type 1 Gaucher disease. The application of combined methods in molecular biology allowed us to analyse the p.Asn 370 Ser mutation. The affected individual activity is very low. First, we have to used the enzymatic digestion method. Then, we have to identified the mutation by the refractory mutation system technique using specific primers for the p.Asn 370 Ser mutation. These analyses are supplemented by the direct sequencing in order to seek and confirm this mutation. Finally, the absence of the 55 pb deletion in exon 9 among corroborated the presence of the homozygous genotype of this p.Asn 370 Ser in the patient DNA.

Brox, a novel farnesylated Bro1 domain‐containing protein that associates with charged multivesicular body protein 4 (CHMP4)

Human Brox is a newly identified 46 kDa protein that has a Bro1 domain-like sequence and a C-terminal thioester-linkage site of isoprenoid lipid (CAAX motif) (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid). Mammalian Alix and its yeast ortholog, Bro1, are known to associate with charged multivesicular body protein 4 (CHMP4), a component of endosomal sorting complex required for transport III, via their Bro1 domains and to play roles in sorting of ubiquitinated cargoes. We investigated whether Brox has an authentic Bro1 domain on the basis of its capacity for interacting with CHMP4s. Both Strep Tactin binding sequence (Strep)-tagged wild-type Brox (Strep-Brox(WT)) and Strep-tagged farnesylation-defective mutant (Cys-->Ser mutation; Strep-Brox(C408S)) pulled down FLAG-tagged CHMP4b that was coexpressed in HEK293 cells. Treatment of cells with a farnesyltransferase inhibitor, FTI-277, caused an electrophoretic mobility shift of Strep-Brox(WT), and the mobility coincided with that of Strep-Brox(C408S). The inhibitor also caused a mobility shift of endogenous Brox detected by western blotting using polyclonal antibodies to Brox, suggesting farnesylation of Brox in vivo. Fluorescence microscopic analyses revealed that Strep-Brox(WT) exhibited accumulation in the perinuclear area and caused a punctate pattern of FLAG-CHMP4b that was constitutively expressed in HEK293 cells. On the other hand, Strep-Brox(C408S) showed a diffuse pattern throughout the cell, including the nucleus, and did not cause accumulation of FLAG-CHMP4b. Fluorescent signals of monomeric green fluorescent protein (mGFP)-fused Brox(WT) merged partly with those of Golgi markers and with those of abnormal endosomes induced by overexpression of a dominant negative mutant of AAA type ATPase SKD1/Vps4B in HeLa cells, but such colocalization was less efficient for mGFP-Brox(C408S). These results suggest a physiological significance of farnesylation of Brox in its subcellular distribution and efficient interaction with CHMP4s in vivo.

Mutation adjacent to the active site tyrosine can enhance DNA cleavage and cell killing by the TOPRIM Gly to Ser mutant of bacterial topoisomerase I

The TOPRIM DXDXXG residues of type IA and II topoisomerases are involved in Mg(II) binding and the cleavage-rejoining of DNA. Mutation of the strictly conserved glycine to serine in Yersinia pestis and Escherichia coli topoisomerase I results in bacterial cell killing due to inhibition of DNA religation after DNA cleavage. In this study, all other substitutions at the TOPRIM glycine of Y. pestis topoisomerase I were examined. While the Gly to Ala substitution allowed both DNA cleavage and religation, other mutations abolished DNA cleavage. DNA cleavage activity retained by the Gly to Ser mutant could be significantly enhanced by a second mutation of the methionine residue adjacent to the active site tyrosine. Induction of mutant topoisomerase with both the TOPRIM glycine and active site region methionine mutations resulted in up to 40-fold higher cell killing rate when compared with the single TOPRIM Gly to Ser mutant. Bacterial type IA topoisomerases are potential targets for discovery of novel antibiotics. These results suggest that compounds that interact simultaneously with the TOPRIM motif and the molecular surface around the active site tyrosine could be highly efficient topoisomerase poisons through both enhancement of DNA cleavage and inhibition of DNA rejoining.

Glycosaminoglycans Facilitate Procathepsin B Activation through Disruption of Propeptide-Mature Enzyme Interactions

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B. We show that naturally occurring GAGs such as chondroitin sulfates and heparin, as well as the synthetic analog dextran sulfate, accelerate the processing in a concentration-dependent manner. Heparin oligosaccharides down to the size of tetrasaccharides were efficient in accelerating the procathepsin B processing, whereas disaccharides were without effect. Further, the ability of the GAGs to accelerate procathepsin B processing was sensitive to increasing NaCl concentrations, indicating that electrostatic interaction between the GAGs and procathepsin B are operative in the accelerating effect. Also the processing of the catalytic procathepsin B mutant by wild type cathepsin B was enhanced in the presence of GAGs, suggesting that GAGs induce a conformational change in procathepsin B, converting it into a better substrate. Site-directed mutagenesis showed that His(28), Lys(39), and Arg(40), located within the procathepsin B propeptide, have significant roles in the acceleration of procathepsin B activation induced by short GAGs. Because procathepsin B and GAGs often co-localize in vivo, we propose that GAGs may play a physiological role in the activation of procathepsin B.

Requirement of Left-Handed Glycine Residue for High Stability of the Tk-Subtilisin Propeptide as Revealed by Mutational and Crystallographic Analyses

Tk-subtilisin [the mature domain of Pro-Tk-subtilisin in active form (Gly70-Gly398)] from the hyperthermophilic archaeon Thermococcus kodakaraensis is matured from Pro-Tk-subtilisin [a subtilisin homologue from T. kodakaraensis in pro form (Gly1-Gly398)] upon autoprocessing and degradation of propeptide. Pro-Tk-subtilisin is characterized by extremely slow maturation at mild temperatures, but this maturation rate is greatly increased by a single Gly56 → Ser mutation in the propeptide region. To analyze the role of Gly56, which assumes a left-handed conformation, Pro-Tk-subtilisin variants with complete amino acid substitutions at Gly56 were constructed. A comparison of their halo-forming activities suggests that all variants, except for Pro-G56W [Pro-G56X, Pro-Tk-subtilisin with Gly56 → X mutation (X = any amino acid)], mature faster than WT. Pro-G56W and Pro-G56E with the lowest and highest maturation rates, respectively, among 19 variants, as well as WT and Pro-G56S, were overproduced, purified, and characterized. SDS-PAGE analyses and Tk-subtilisin activity assay indicated that their maturation rates increased in the order WT ≤ Pro-G56W < Pro-G56S < Pro-G56E. The propeptides of these variants were also overproduced, purified, and characterized. The stability and inhibitory potency of these propeptides decreased in the order Tk-propeptide [propeptide of Tk-subtilisin (Gly1-Leu69)] ≥ G56W-propeptide > G56S-propeptide > G56E-propeptide, indicating that they are inversely correlated with the maturation rates of Pro7-Tk-subtilisin and its derivatives. The crystal structures of these propeptides determined in complex with S324A-subtilisin indicate that the conformation of the propeptide is altered by the mutation, such that nonglycine residues at position 56 assume a right-handed conformation and hydrophobic interactions at the core region decrease. These results indicate that Gly56 is required in stabilizing the propeptide fold. Stabilization of this fold leads to strong binding of Tk-propeptide to Tk-subtilisin, high resistance of Tk-propeptide to proteolytic degradation, and slow maturation of Pro-Tk-subtilisin.

A Multinuclear Copper(I) Cluster Forms the Dimerization Interface in Copper-Loaded Human Copper Chaperone for Superoxide Dismutase

Copper binding and X-ray aborption spectroscopy studies are reported on untagged human CCS (hCCS; CCS = copper chaperone for superoxide dismutase) isolated using an intein self-cleaving vector and on single and double Cys to Ala mutants of the hCCS MTCQSC and CSC motifs of domains 1 (D1) and 3 (D3), respectively. The results on the wild-type protein confirmed earlier findings on the CCS-MBP (maltose binding protein) constructs, namely, that Cu(I) coordinates to the CXC motif, forming a cluster at the interface of two D3 polypeptides. In contrast to the single Cys to Ser mutations of the CCS-MBP protein (Stasser, J. P., Eisses, J. F., Barry, A. N., Kaplan, J. H., and Blackburn, N. J. (2005) Biochemistry 44, 3143-3152), single Cys to Ala mutations in D3 were sufficient to eliminate cluster formation and significantly reduce CCS activity. Analysis of the intensity of the Cu-Cu cluster interaction in C244A, C246A, and C244/246A variants suggested that the nuclearity of the cluster was greater than 2 and was most consistent with a Cu4S6 adamantane-type species. The relationship among cluster formation, oligomerization, and metal loading was evaluated. The results support a model in which Cu(I) binding converts the apo dimer with a D2-D2 interface to a new dimer connected by cluster formation at two D3 CSC motifs. The predominance of dimer over tetramer in the cluster-containing species strongly suggests that the D2 dimer interface remains open and available for sequestering an SOD1 monomer. This work implicates the copper cluster in the reactive form and adds detail to the cluster nuclearity and how copper loading affects the oligomerization states and reactivity of CCS for its partner SOD1.

Small Ubiquitin-like Modifier Modification Regulates the DNA Binding Activity of Glial Cell Missing Drosophila Homolog a

Glial cell missing Drosophila homolog a (GCMa) is an essential transcription factor for placental development, which controls the differentiation of the syncytiotrophoblast layer. Although the activity of GCMa can be post-translationally regulated by protein phosphorylation, ubiquitination, and acetylation, it is unknown whether GCMa activity can be regulated by sumoylation. In this report, we investigated the role of sumoylation in the regulation of GCMa activity. We demonstrated that Ubc9, the E2 component of the sumoylation machinery, specifically interacts with the N-terminal domain of GCMa and promotes GCMa sumoylation on lysine 156. Moreover, GCMa-mediated transcriptional activation was repressed by sumoylation but was enhanced in the presence of the SUMO-specific protease, SENP1. The repressive effect of sumoylation on GCMa transcriptional activity was attributed to decreased DNA binding activity of GCMa. Furthermore, structural analysis revealed a steric clash between the SUMO1 moiety of sumoylated GCMa and the DNA-binding surfaces of GCMa, which may destabilize the interaction between GCMa and its cognate DNA sequence. Our study demonstrates that GCMa is a new sumoylation substrate and its activity is down-regulated by sumoylation.

Differential desensitization of dopamine D 2 receptor isoforms by protein kinase C: The importance of receptor phosphorylation and pseudosubstrate sites

Altered regulation of dopamine D 2 receptors is implicated in addiction, schizophrenia and movement disorders, as well as lactotroph growth and regulation. Dopamine D 2S and dopamine D 2L receptors are alternately-spliced variants that differ by 29 amino acids in the third intracellular (i3) domain and display different sensitivity to desensitization by protein kinase C (PKC). In the present studies we determined the specific phosphorylation sites on the dopamine D 2S receptor that confer PKC-mediated desensitization. In dopamine D 2L receptors, we identified a PKC pseudosubstrate site responsible for the relative insensitivity of the receptor to PKC-induced uncoupling. In transiently transfected Ltk − fibroblast cells, 2-min preactivation of PKC with 12- O-tetradecanoyl 4β-phorbol 13α-acetate (TPA) completely inhibited calcium mobilization induced by the dopamine D 2S receptor, but not the dopamine D 2L variant. Point mutation of i3 PKC sites Ser228/229Gly rendered the dopamine D 2S receptor resistant to PKC action, with lesser effects of other Ser and Thr mutations. Inactivation of the PKC pseudosubstrate motif in the dopamine D 2L receptor sensitized the receptor to PKC, and this was reversed by mutation of i3 PKC sites Ser228/229. A phospho-specific antibody generated against phospho-Ser228/229 demonstrated PKC-induced phosphorylation at these sites of dopamine D 2S, but not D 2L receptors, in Ltk − cells. Conversely, the pseudosubstrate dopamine D 2L receptor mutant displayed PKC-induced phosphorylation at Ser228/229, which was abolished when these sites were mutated. Similar phosphorylation results were observed using GH4 cells stably transfected with dopamine D 2 receptors and mutants. Thus the relative location of phosphorylation and pseudosubstrate sites provides an important determinant substrate sensitivity to PKC.

Characterization and Structure of a Zn2+ and [2Fe-2S]-containing Copper Chaperone from Archaeoglobus fulgidus

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 A resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.

Molecular characterisation of resistance to ALS‐inhibiting herbicides in Hordeum leporinum biotypes

The acetolactate synthase (ALS)-inhibiting herbicide sulfosulfuron is registered in Australia for the selective control of Hordeum leporinum Link. in wheat crops. This herbicide failed to control H. leporinum on two farms in Western Australia on its first use. This study aimed to determine the level of resistance of three H. leporinum biotypes, identify the biochemical and molecular basis and develop molecular markers for diagnostic analysis of the resistance. Dose-response studies revealed very high level (>340-fold) resistance to the sulfonylurea herbicides sulfosulfuron and sulfometuron. In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to herbicide inhibition. This altered ALS sensitivity in the resistant biotypes was found to be due to a mutation in the ALS gene resulting in amino acid proline to serine substitution at position 197. In addition, two- to threefold higher ALS activities were consistently found in the resistant biotypes, compared with the known susceptible biotype. Two cleaved amplified polymorphic sequence (CAPS) markers were developed for diagnostic testing of the resistant populations. This study established the first documented case of evolved ALS inhibitor resistance in H. leporinum and revealed that the molecular basis of resistance is due to a Pro to Ser mutation in the ALS gene.

Protein Kinase Cζ Abrogates the Proapoptotic Function of Bax through Phosphorylation

Protein kinase Czeta (PKCzeta) is an atypical PKC isoform that plays an important role in supporting cell survival but the mechanism(s) involved is not fully understood. Bax is a major member of the Bcl-2 family that is required for apoptotic cell death. Because Bax is extensively co-expressed with PKCzeta in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells, it is possible that Bax may act as the downstream target of PKCzeta in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKCzeta activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKCzeta specific inhibitor (a myristoylated PKCzeta pseudosubstrate peptide). Purified, active PKCzeta directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKCzeta mutant results in Bax phosphorylation at serine 184. PKCzeta co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKCzeta by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKCzeta mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKCzeta can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKCzeta may function as a physiological Bax kinase to directly phosphorylate and interact with Bax, which leads to sequestration of Bax in cytoplasm and abrogation of the proapoptotic function of Bax.

Nox1 Redox Signaling Mediates Oncogenic Ras-induced Disruption of Stress Fibers and Focal Adhesions by Down-regulating Rho

Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Nox1 small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1-generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes.

An affinity-based fluorescence polarization assay for protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild-type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate “false” positives due to modification of the active site Cys that destroy the phosphatase activity.

Impact of in vitro assembly defects on in vivo function of the phage P22 portal

The podovirus P22, which infects O-antigen strains of Salmonella, incorporates a dsDNA translocating channel (portal dodecamer) at a unique vertex of the icosahedral capsid. The portal subunit (gp1, 82.7 kDa) exhibits multiple S–H⋯X hydrogen bonding states for cysteines 153, 173, 283 and 516 and these interactions are strongly perturbed by portal ring formation. Here, we analyze in vivo activities of wild type (wt) and Cys → Ser mutant portals, demonstrate that in vivo activity is correlated with in vitro assembly kinetics, and suggest mechanistic bases for the observed assembly defects. The C283S portal protein, which assembles into rings at about half the rate of wt, exhibits significantly diminished infectivity (∼ 50% of wt) and manifests its defect prior to DNA packaging, most likely at the stage of procapsid assembly. Conversely, the C516S mutant, which assembles at twice the rate of wt, is more severely deficient in vivo (∼ 20% of wt) and manifests its defect subsequent to capsid maturation and DNA packaging. Both C153S and C173S portals function at levels close to wt. The results suggest that C283S and C516S mutations may be exploited for improved characterization of the folding and assembly pathway of P22 portal protein.

Selenoprotein H Is a Nucleolar Thioredoxin-like Protein with a Unique Expression Pattern

The human selenoproteome consists of 25 known selenoproteins, but functions of many of these proteins are not known. Selenoprotein H (SelH) is a recently discovered 14-kDa mammalian protein with no sequence homology to functionally characterized proteins. By sensitive sequence and structure analyses, we identified SelH as a thioredoxin fold-like protein in which a conserved CXXU motif (cysteine separated by two other residues from selenocysteine) corresponds to the CXXC motif in thioredoxins. These data suggest a redox function of SelH. Indeed, a recombinant SelH shows significant glutathione peroxidase activity. In addition, SelH has a conserved RKRK motif in the N-terminal sequence. We cloned wild-type and cysteine mutant forms of SelH either upstream or downstream of green fluorescent protein (GFP) and localized this fusion protein to the nucleus in transfected mammalian cells, whereas mutations in the RKRK motif resulted in the cytosolic protein. Interestingly, the full-length SelH-GFP fusion protein localized specifically to nucleoli, whereas the N-terminal sequence of SelH fused to GFP had a diffuse nucleoplasm location. Northern blot analyses revealed low expression levels of SelH mRNA in various mouse tissues, but it was elevated in the early stages of embryonic development. In addition, SelH mRNA was overexpressed in human prostate cancer LNCaP and mouse lung cancer LCC1 cells. Down-regulation of SelH by RNA interference made LCC1 cells more sensitive to hydrogen peroxide but not to other peroxides tested. Overall, these data establish SelH as a novel nucleolar oxidoreductase and suggest that some functions in this compartment are regulated by redox and dependent on the trace element selenium.