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PubMed2000

bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells.

bcl-2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over-expression of bcl-2 in a human breast-cancer cell line (MCF7(ADR)) enhances its tumorigenicity and metastatic potential by inducing metastasis-associated properties such as increased secretion of the matrix metalloproteinase-9 (mmp-9). In the present study, we investigated the effect of bcl-2 over-expression on the activity of the transcription factor NF-kappaB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over-expression of bcl-2 in the MCF7(ADR) cell line, enhances NF-kappaB-dependent transcriptional activity. Mobility-shift analysis revealed an increase of NF-kappaB DNA-binding in bcl-2-over-expressing clones that correlated with lower levels of the NF-kappaB cytoplasmic inhibitor IkappaBalpha. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl-2 with bax, or deletion of the N-terminal BH4 domain, completely eliminate the ability of this molecule to up-regulate NF-kappaB-dependent transactivation. Since mmp-9 is a NF-kappaB-regulated gene, we also investigated whether bcl-2 over-expression up-regulated mmp-9 transcription. We found that induction of mmp-9 mRNA correlates with the activation of an mmp-9-promoter-reporter-gene construct in transient transfection assay, and a mutation of the (-600)mmp-9-NF-kappaB binding element abolishes this effect. The overall data indicate that bcl-2-mediated regulation of NF-kappaB-transcription-factor activity may represent an important mechanism for the promotion of malignant behavior in MCF-7(ADR) cells.

Binding Sites↗
PubMed1995

Identification by mutation of the tyrosine residues in the insulin receptor substrate-1 affecting association with the tyrosine phosphatase 2C and phosphatidylinositol 3-kinase.

The insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on several tyrosine residues by the activated insulin receptor. Phosphorylated IRS-1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins involved in insulin signaling. These include in vivo the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3-K) and the phosphotyrosine phosphatase-2C (PTP2C). In this report, we examined which tyrosine residues of IRS-1 are required for the interactions of IRS-1 with PI3-K and PTP2C. To address this issue, we constructed different rat IRS-1 mutants containing mutations in the tyrosine residues that interact with the SH2 domains of PI3-K and PTP2C in vitro. Each of the IRS-1 mutants obtained have been transiently expressed in 293 EBNA cells to study their ability to interact with PI3-K and PTP2C in vivo. Our results demonstrate that mutation of tyrosine 608 affects the PI3-K activity associated with IRS-1, suggesting that this tyrosine is likely to be a principal site of interaction with the SH2 domains of p85 in response to insulin. Furthermore, we found that mutation of tyrosines 1172 and 1222 totally prevents the insulin-induced association of IRS-1 with the SH2 domains of PTP2C, demonstrating that both tyrosines 1172 and 1222 are key elements in the binding sites for the SH2 domains of PTP2C. Finally, we found that the ability of purified PTP2C to dephosphorylate IRS-1 is dependent on the association of PTP2C with phosphorylated IRS-1.

Animals↗
PubMed1993

Carcinogen-induced diploid hepatocytes: sensitive target cells for transformation by mutated c-Ha-ras oncogene.

Sequential treatment of partially (two-thirds) hepatectomized rats with diethylnitrosamine and 2-acetylaminofluorene induces the emergence of diploid hepatocytes in rat liver. These carcinogen-induced diploid cell populations are thought to contain the progenitors of hepatocellular carcinoma (HCC), i.e., initiated, cells. In the study presented here, we addressed the question of whether putative mutations in carcinogen-induced diploid hepatocytes can cooperate with activated oncogenes in the process of transformation in vitro. Both carcinogenesis in vivo and transformation in vitro have been shown to be multistep processes requiring at least two independent transforming events. Diploid and polyploid rat hepatocytes were isolated by centrifugal elutriation. The purity of the elutriated fractions was 88 +/- 3% in the diploid fraction and 84 +/- 3% in the polyploid fraction. Hepatocytes from both the elutriated cell fractions and, for comparison, hepatocytes from untreated rats were transfected by electroporation with oncogene expression vectors containing the mutated human T24 c-Ha-ras gene and of the N-myc gene. Transient expression of transfected DNA was similar in both hepatocyte populations. No cell lines could be established by using the N-myc vector. In contrast, the carcinogen-induced diploid hepatocytes, but not polyploid hepatocytes, could be converted by transfection with the ras vector into permanent anchorage-independent growing cell lines with hepatocyte-like morphology and differentiation. These cell lines expressed the myc proto-oncogene and transforming growth factor-alpha constitutively. Thus, carcinogen-induced diploid hepatocytes are sensitive to transformation by the ras oncogene, suggesting cooperation between putative preexisting mutations in the diploid cells and the ras oncogene product in hepatocellular transformation.

2-Acetylaminofluorene↗
PubMed2000

Mutations selected in rotavirus enterotoxin NSP4 depend on the context of its expression.

The rotavirus NSP4 protein is cytotoxic when transiently expressed in cells and is capable of inducing secretory diarrhea in neonatal mice. NSP4 consists of 175 amino acids, and sequences important for its toxic effects have been mapped to the carboxy-terminal half of the protein. In this report, we compared NSP4-encoding nucleotide sequences recovered from cell lines engineered to express NSP4 from human rotavirus strain Wa with NSP4 sequences recovered from cells persistently infected with either Wa or simian rotavirus strain SA11. In cells stably transfected with Wa NSP4, we found that proline(138) was changed to either serine or threonine. However, in cells persistently infected with SA11, we found that phenylalanine(33) was changed to leucine, and in cells persistently infected with Wa, no changes were observed in NSP4. Expression of Wa NSP4 in Caco-2 cells resulted in increased cell-doubling times and decreased cell viability in comparison to cells expressing NSP4-serine(138) or NSP4-threonine(138). This result suggests that sequence polymorphism at residue 138 in Wa NSP4 influences the cytotoxicity of the protein. Therefore, mutations in the carboxy-terminal half of NSP4 are selected when NSP4 is expressed in cells in the absence of other viral proteins, but not in the context of viral replication. These findings suggest that cytotoxic functions of NSP4 are not operant during natural rotavirus infection.

Amino Acid Substitution↗
PubMed2002

Effects of adeno-associated virus-vectored ciliary neurotrophic factor on retinal structure and function in mice with a P216L rds/peripherin mutation.

Past studies have shown that acute administration of ciliary neurotrophic factor (CNTF) can prolong the survival of retinal photoreceptor cells that have undergone phototoxic injury or that express gene mutations. Adenovirus-vectored CNTF has also been effective but for all of these treatments, the effect has been transient. On the other hand, adeno-associated virus-vectored minigenes offer considerable promise for long-term survival. The authors sought to provide long-term, CNTF-based protection of mouse photoreceptors expressing a dominant-negative point mutation in the rds gene by using recombinant adeno-associated virus (rAAV) to deliver minigenes that code for a secreted form of CNTF.Secreted CNTF, under control of a cytomegalovirus (CMV) or chick beta actin (CBA) promoter provided long-term, panretinal rescue of photoreceptors following single injections of rAAV vectors into the subretinal compartment. Rescue was much less effective and less reproducible when the vectors were placed in the vitreous compartment. However, there were unexpected side effects that appeared to be dose-related. One side effect was a change in rod photoreceptor nucleus phenotype, featuring an increase in euchromatin and an increase in nuclear size following subretinal injections but not intravitreal injections. These nuclear changes were panretinal when the putatively stronger CBA promoter was used but not panretinal when the CMV promoter was used. In the latter case, the nuclear changes were much more pronounced at the site of injection. Thus, chronic hyperstimulation of retinal cells with CNTF may up-regulate gene expression in photoreceptors. Based on current knowledge of retinal cell targets for CNTF, this effect may be indirect and may not represent direct stimulation of photoreceptors by CNTF.A second side effect was a paradoxical decrease in scotopic a- and b-wave amplitudes and a decrease in photopic b-wave amplitudes in the injected, rescued retina when compared to its contralateral, uninjected counterpart, in spite of the fact that these retinas had more photoreceptors than their untreated mates. The basis for these decreased ERG amplitudes may be related to changes in gene expression. The mechanisms for these side effects and proper doses of CNTF administration should be determined before human clinical trials are considered for the amelioration of inherited retinal degenerations with CNTF.

Adenoviridae↗
PubMedDate not supplied

NF-kappaB is essential for induction of pro-inflammatory cytokine genes by filarial parasitic sheath proteins.

The transcription factor NF-kappaB plays critical roles in immune and inflammatory responses. Here we show that filarial parasitic sheath proteins cause activation of NF-kappaB in the airway epithelial HEp-2 cell line. This activation was transient and saturable, and involved degradation of the cytoplasmic inhibitor protein IkappaBalpha. Stable expression of IkappaBalpha mutated at Ser32 and Ser36 to Ala caused inhibition of NF-kappaB activation, indicating that this activation involves the IkappaB kinase-mediated pathway. Moreover, while it did not influence the HEp-2 cell survival, selective blockade of NF-kappaB activation resulted in inhibition of the expression and the secretion of pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-6 and interleukin-8. Thus, initial transient activation of NF-kappaB resulted in profound and long-term effects on epithelial cell responses to filarial parasitic proteins. These findings implicate an important role for NF-kappaB in orchestrating inflammatory reactions associated with tropical pulmonary eosinophilia.

Animals↗
PubMed2000

The PDZ protein TIP-1 interacts with the Rho effector rhotekin and is involved in Rho signaling to the serum response element.

The human T-cell lymphotrophic virus, type 1 Tax protein can interact via its C terminus with various proteins including a PDZ domain. In this work, one of them, TIP-1, is characterized as a cytoplasmic 14-kDa protein mainly corresponding to one PDZ domain. A two-hybrid screen performed with TIP-1 as bait showed that it interacts with the human homologue of rhotekin that was previously identified in mice as a Rho effector. Both human and mouse rhotekins exhibit at their C termini the sequence QSPV-COOH that matches the X(S/T)XV-COOH consensus known for proteins recognizing PDZ domains. Mutation of the serine and valine residues to alanine impairs interaction of rhotekin with TIP-1. Transient expression experiments with a reporter construct including the c-Fos serum response element (SRE) showed that coexpression of TIP-1 with the constitutively active RhoA.V14 mutant and human rhotekin caused a strong activation of the SRE. A negative mutant of Rho, RhoA.N19, was unable to cooperate with TIP-1 and rhotekin. The positive effect of TIP-1 was also lost when the C terminus of rhotekin was mutated. These data show that the complex of active Rho with its effector rhotekin bound to TIP-1 produces in the cytoplasm a signal that triggers strong activation of the SRE.

Amino Acid Sequence↗
PubMed1994

Identification of a point mutation in the thyrotropin receptor of the hyt/hyt hypothyroid mouse.

The hyt/hyt hypothyroid mouse has an autosomal recessive, fetal-onset, severe hypothyroidism related to TSH hyporesponsiveness and associated with elevated TSH. Our previous work has suggested that the hypothyroidism and TSH hyporesponsiveness may result from a mutation in the hyt/hyt TSH receptor (TSHr) of the thyroid gland. Based on DNA sequencing of the entire coding region of the TSHr gene from the wild-type BALB/cBY +/+ mouse, the +/+ TSHr is 92% and 94% identical at the nucleotide and amino acid residue levels, respectively, compared to the rat TSHr gene. The coding region of the hyt/hyt TSHr, compared to that of the +/+ TSHr, has a single base change, CCG to CTG, at nucleotide position 1666, which leads to the replacement of a highly conserved proline at amino acid position 556 with a leucine in transmembrane domain IV. This mutation was introduced by site-directed mutagenesis into the wild-type human TSHr and transiently expressed in COS-7 cells. Although the size and abundance of the mutant TSHr mRNA suggested that there was no effect on the nature of the mRNA, TSH binding and the response to TSH in transfected cells were abolished. Further studies are necessary to clarify how the Pro to Leu replacement interferes with receptor expression on the cell surface or influences TSH binding. These functional consequences of the mutation appear to account for the observed TSH hyporesponsiveness and hypothyroidism in the hyt/hyt mouse.

Amino Acid Sequence↗
PubMed2005

A C-terminal skeletal muscle sodium channel mutation associated with myotonia disrupts fast inactivation.

Missense mutations in the skeletal muscle sodium channel alpha-subunit gene (SCN4A) are associated with a group of clinically overlapping diseases caused by alterations in the excitability of the sarcolemma. Sodium channel defects may increase excitability and cause myotonic stiffness or may render fibres transiently inexcitable to produce periodic paralysis. A patient with cold-aggravated myotonia did not harbour any of the common SCN4A mutations. We therefore screened all 24 exons by denaturing high-performance liquid chromatography, followed by direct sequencing. Two novel missense changes were found with predicted amino acid substitutions: T323M in the DIS5-S6 loop and F1705I in the intracellular C-terminus. The functional impact of these substitutions was assessed by recording whole-cell Na+ currents from transiently transfected HEK293 cells. T323M currents were indistinguishable from wild-type (WT). Fast inactivation was impaired for F1705I channels, as demonstrated by an 8.6-mV rightwards shift in voltage dependence and a two-fold slowing in the rate of inactivation. Recovery from fast inactivation was not altered, nor was there an increase in the persistent current after a 50- ms depolarization. Activation and slow inactivation were not appreciably affected. These data suggest that T323M is a benign polymorphism, whereas F1705I results in fast inactivation defects, which are often observed for myotonia. This is the first example of a C-terminal mutation in SCN4A associated with human disease. Like the cardiac disorders (long QT syndrome type 3 or Brugada syndrome) and generalized epilepsy with febrile seizures plus (GEFS+) associated with C-terminal mutations in other NaV channels, the primary effect of F1705I was a partial disruption of fast inactivation.

Amino Acid Sequence↗
PubMed2006

Novel approaches in the treatment of systemic mastocytosis.

In the absence of curative options, therapy for aggressive forms of systemic mastocytosis (SM) has relied in the use of cytoreductive agents, mainly interferon-alpha (IFN-alpha) and cladribine. However, responses are transient and only occur in a subset of patients. Gain-of-function mutations at codon 816 of the KIT protooncogene lead to constitutively active Kit receptor molecules, which are central to the pathogenesis of SM. Recent advances in the understanding of the molecular underpinnings of SM have led to the development of small molecules targeting mutant Kit tyrosine kinase isoforms that significantly have widened the range of therapeutic options for patients with SM. Some of these promising agents, such as dasatinib, AMN107, and PKC412, currently are under investigation in clinical trials whereas, others are at different stages of preclinical development. In addition, monoclonal antibodies directed to neoplastic mast cell-restricted surface antigens constitute a viable option for the treatment of SM that warrants further investigation.

Antibodies, Monoclonal↗
PubMed2001

Involving AP-2 transcription factor in connexin 26 up-regulation during pregnancy and lactation.

Gap junction connexin 26 (Cx26) is up-regulated in mammary epithelial cells during pregnancy and lactation. To understand the transcriptional regulation of Cx26, we identified a protected DNase I footprint region (-140 to -113) in the rat Cx26 promoter. This rCx26 Promoter Footprinting Region, or CPFR, contains an Sp binding site (CCGCCC) overlapping with an AP-2 binding site (GCCCGCGGC), and is evolutionarily conserved. Nuclear extracts from rat mammary glands and human MCF-10 mammary epithelial cells formed protein-DNA complexes with the labeled CPFR probe in the electrophoretic mobility shift assay (EMSA), and these complexes were markedly enhanced during pregnancy and lactation. Antibody supershift analysis further identified the presence of Sp1, Sp3, and AP-2 in these binding complexes. Human mammary epithelial MCF-10A and MCF-12A cells were transiently transfected with chimeric mutant rCx26 promoter/luciferase reporter constructs, and luciferase activities measured. Mutations along the CPFR fragment drastically reduced the promoter activity, specially at the Sp/AP-2 overlapping site. Cotransfection of AP-2 with rCx26 promoter/reporter constructs into MCF-10 cells markedly induced the reporter activity. These data infer that AP-2, along with previously reported Sp transcription factors, is involved in the up-regulation of Cx26 gene during pregnancy and lactation.

Animals↗
PubMed2005

Different levels of ovine interferon-tau gene expressions are regulated through the short promoter region including Ets-2 binding site.

Regulation of interferon-tau (IFNtau) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, has not been fully elucidated. Among more than 10 ovine IFNtau (oIFNtau) gene sequences characterized, approximately 75% of oIFNtau transcripts expressed in utero is derived from oIFNtau-o10 gene and amounts of transcripts from other oIFNtau genes such as oIFNtau-o8 or oIFNtau-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNtau-o10 and oIFNtau-o8/-o2 genes was due to differences in the proximal promoter regions of these oIFNtau genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 5'-upstream regions of these oIFNtau genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNtau-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNtau-o10 gene inserted into the oIFNtau-o8/-o2 gene-reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNtau-o8/o2-reporter construct failed to increase the degree of transactivation. Rather than this 6 bp sequence, a 22 bp sequence of the proximal promoter region, including the Ets-2 binding site, of the oIFNtau-o10 gene was required for oIFNtau-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNtau-o10 and oIFNtau-o8/o2 genes differed. These results suggest that the short promoter region including the Ets-2 binding site, not the Ets-2 binding region itself, may determine different levels of oIFNtau gene expressions seen in utero.

Animals↗
PubMed1998

Identification of single amino acids in the human papillomavirus 11 E2 protein critical for the transactivation or replication functions.

The papillomavirus E2 protein is required for viral transcriptional regulation and replication. The E2 protein has a modular structure with two highly conserved domains, a sequence-specific DNA-binding and dimerization domain and a conserved N-terminus which is important for transcriptional transactivation, replication, and interaction with the E1 protein to determine which specific amino acids or regions in the N-terminus were important for the replication or transactivation functions. Single amino acid substitutions were created at highly conserved, highly charged amino acids in the HPV 11 E2 N-terminus. Each amino acid was mutated to a nonpolar alanine residue or a similarly charged amino acid. The mutated E2 proteins were analyzed for their abilities to support transcriptional transactivation and transient DNA replication and to enhance binding of E1 to the origin of replication. Single amino acid substitutions were identified which were defective for either the replication or transactivation functions, which demonstrated that the replication and transactivation functions within the N-terminus are separable. In several cases different amino acid substitutions at the same site had variable effects on transcription or replication, highlighting the importance of hydrophobic interactions or side chain structure at each site. The replication function appeared to correlate with the ability of E2 to enhance binding of E1 to the origin of replication though these studies also suggest that other functions performed by the E2 protein may be important for replication.

Alanine↗
PubMed2003

WT1 expression does not disrupt myogenic differentiation in C2C12 murine myoblasts or in human rhabdomyosarcoma.

WT1 encodes a tissue-specific transcription factor important in early mesenchymal differentiation. Altered expression or mutation of WT1 occurs in malignancies derived from such tissues. These include Wilms tumour, a paediatric kidney cancer that may show heterologous differentiation into primitive skeletal muscle, especially in tumours with WT1 mutation. A putative role for WT1 in inhibiting myogenesis has been suggested by transient transfection of C(2)C(12) myoblasts. However, using a more robust model of stable transfectants of C(2)C(12) expressing inducible WT1 isoforms, we found no inhibition of myogenic differentiation. We also investigated a possible role for WT1 in the disrupted myogenesis seen in rhabdomyosarcoma, a paediatric cancer resembling foetal skeletal muscle. WT1 expression levels measured by quantitative real-time reverse transcription polymerase chain reaction were low or absent in those tumours with a PAX-FKHR fusion gene characteristic of the alveolar subtype, and were higher in cases lacking these fusion genes. Overall, there was a weak positive correlation between expression of myogenic differentiation marker genes and WT1 levels. We conclude that expression of WT1 in C(2)C(12) cells and in rhabdomyosarcoma does not inhibit myogenic differentiation.

Animals↗
PubMed1994

Alpha 1-proteinase inhibitor variant T345R. Influence of P14 residue on substrate and inhibitory pathways.

To test whether the presence of charged residues at position P14 of the reactive center region of noninhibitory members of the serpin family of protein proteinase inhibitors is responsible for their lack of proteinase inhibitory properties, we expressed a variant of the alpha 1-proteinase inhibitor (alpha 1-PI) with arginine substituted for threonine at this position (T345R) and characterized its functional properties. Although the T345R variant reacted with proteinases principally as a substrate, it was still capable of forming stable complexes with the three serine proteinases examined, human neutrophil elastase (HNE), porcine pancreatic elastase (PPE), and trypsin. The fraction of T345R alpha 1-PI that formed a complex with proteinase was quantitated by autoradiography of SDS gels of the variant incubated with 125I-labeled proteinase. The stoichiometry of inhibition (S.I.) (number of mol of alpha 1-PI required to completely inhibit 1 mol of proteinase), which was 1 for both plasma alpha 1-PI and wild-type recombinant alpha 1-PI interacting with each of the proteinases, was very much greater than 1 for T345R variant alpha 1-PI. Values of 9.5, 45, and about 70 were estimated for variant alpha 1-PI inhibition of trypsin, HNE, and PPE, respectively. An inverse relationship between the apparent second-order rate constant and the S.I. for inhibition of PPE by T345R alpha 1-PI suggested that the mutation did not affect the rate-determining step of formation of a transient intermediate complex. Following cleavage of the reactive center loop, there was a large increase in protein stability and changes in the CD spectrum, both consistent with insertion of the reactive center loop into beta-sheet A. This behavior is similar to that of wild-type alpha 1-PI. We conclude that the presence of a charged residue at P14 does not prevent reactive center loop insertion or the functioning of alpha 1-PI as an inhibitor of serine proteinases but does significantly alter the relative rates of the substrate and inhibitory pathways in favor of the former, probably by reducing the rate of the latter reaction.

Animals↗
PubMed1993

Rationally designing the accumulation of a folding intermediate of barnase by protein engineering.

A method for the stabilization of transient folding intermediates is presented. Barnase folds and unfolds via such an intermediate. Mutations that destabilize the folded state relative to the folding intermediate had been previously identified from the free energy profiles for the unfolding of mutant proteins. It is predicted that the accumulation of such mutations should lead to the intermediate being the most stable species at certain concentrations of denaturant. Mutants were prepared that contained combinations of such mutations. The behavior of these mutants on urea denaturation was studied by probes for tertiary structure (fluorescence, near-UV CD), secondary structure (far-UV CD), and hydrodynamic volume (size-exclusion chromatography). Whereas wild-type shows a two-state transition in all cases, with the same thermodynamic values being found by all probes, some of the mutants show different transitions with different structural probes. On increasing concentration of denaturant, the tertiary structure of these mutants is lost before all the secondary structure and before the protein shows the maximum expanded volume that is characteristic of the unfolded state. These mutants thus accumulate an intermediate state at equilibrium under certain urea concentrations. The intermediate state retains some degree of secondary structure but has a disrupted tertiary structure, and its degree of compactness is intermediate between the folded and the unfolded forms, probably expanding with increasing concentration of denaturant. The accumulation of the intermediate should allow its direct characterization by spectroscopy, especially NMR.

Bacterial Proteins↗
PubMed2001

Modifications of cysteine residues in the solution and membrane-associated conformations of phosphatidylinositol transfer protein have differential effects on lipid transfer activity.

The alpha isoforms of mammalian phosphatidylinositol transfer protein (PITP) contain four conserved Cys residues. In this investigation, a series of thiol-modifying reagents, both alkylating and mixed disulfide-forming, was employed to define the accessibility of these residues and to evaluate their role in protein-mediated intermembrane phospholipid transport. Isolation and analysis of chemically modified peptides and site-directed mutagenesis of each Cys residue to Ala were also performed. Soluble, membrane-associated, and denatured preparations of wild-type and mutant rat PITPs were studied. Under denaturing conditions, all four Cys residues could be detected spectrophotometrically by chemical reaction with 4,4'-dipyridyl disulfide or 5,5'-dithiobis(2-nitrobenzoate). In the native protein, two of the four Cys residues were sensitive to some but not all thiol-modifying reagents, with discrimination based on the charge and hydrophobicity of the reagent and the conformation of the protein. With the soluble conformation of PITP, achieved in the absence of phospholipid vesicles, the surface-exposed Cys(188) was chemically modified without consequence to lipid transfer activity. Cys(188) exhibited an apparent pK(a) of 7.6. The buried Cys(95), which constitutes part of the phospholipid substrate binding site, was covalently modified upon transient association of PITP with a membrane surface. The Cys-to-Ala mutations showed that neither Cys(95) nor Cys(188) was essential for lipid transfer activity. However, chemical modification of Cys(95) resulted in the loss of lipid transfer activity. These results demonstrate that the Cys residues of PITP can be assigned to several different classes of chemical reactivity. Of particular interest is Cys(95), whose sulfhydryl group becomes exposed to modification in the membrane-associated conformation of PITP. Furthermore, the inhibition of PITP activity by thiol-modifying reagents is a result of steric hindrance of phospholipid substrate binding.

Animals↗
PubMed2004

Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants.

All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c(2), the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome c(2) and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.

Cytochromes c2↗