Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation

We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin alpha(IIb)beta(3). In contrast, disruption of each of the disulfide bonds in the two long insertions of the I-like domain predicted to be in close contact with the alpha subunit beta-propeller domain affect the stability of the alpha(IIb)beta(3) heterodimer and inhibit complex-specific mAb binding without affecting the RGD binding capacity of the metal ion-dependent adhesion site-like domain.     

Conserved cysteines in the sialyltransferase sialylmotifs form an essential disulfide bond

The sialyltransferase gene family is comprised of 16 cloned enzymes. All members contain two conserved protein domains, termed the S- and L-sialylmotifs, that participate in substrate binding. Of only six invariant amino acids, two are cysteines, with one found in each sialylmotif. Although the recombinant soluble form of ST6Gal I has six cysteines, quantitative analysis indicated the presence of only one disulfide linkage, and thiol reducing agents dithiothreitol and beta-mercaptoethanol inactivated the enzyme. Analysis of site-directed mutants showed that alanine or serine mutants of invariant Cys(181) or Cys(332) exhibit no detectable activity, either by direct assay or by staining of the transfected cells with Sambucus nigra agglutinin, which recognizes the product NeuAcalpha2,6Galbeta1,4GlcNAc on glycoproteins. In contrast, alanine mutations of charged residues adjacent to either cysteine showed little or no effect on enzyme activity. Immunofluorescence microscopy showed that although the wild type sialyltransferase is properly localized in the Golgi apparatus, the inactive cysteine mutants are retained in the endoplasmic reticulum. The results suggest that the invariant cysteine residues in the L- and S-sialylmotifs participate in the formation of an intradisulfide linkage that is essential for proper conformation and activity of ST6Gal I.     

Direct evidence that two cysteines in the dopamine transporter form a disulfide bond

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.     

Specific integrin alpha and beta chain phosphorylations regulate LFA-1 activation through affinity-dependent and -independent mechanisms

Integrins are adhesion receptors that are crucial to the functions of multicellular organisms. Integrin-mediated adhesion is a complex process that involves both affinity regulation and cytoskeletal coupling, but the molecular mechanisms behind this process have remained incompletely understood. In this study, we report that the phosphorylation of each cytoplasmic domain of the leukocyte function-associated antigen-1 integrin mediates different modes of integrin activation. alpha Chain phosphorylation on Ser1140 is needed for conformational changes in the integrin after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1 (Rap1V12). In contrast, the beta chain Thr758 phosphorylation mediates selective binding to 14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells.     

Two cysteines in each periplasmic domain of the membrane protein DsbB are required for its function in protein disulfide bond formation.

DsbB is a protein component of the pathway that leads to disulfide bond formation in periplasmic proteins of Escherichia coli. Previous studies have led to the hypothesis that DsbB oxidizes the periplasmic protein DsbA, which in turn oxidizes the cysteines in other periplasmic proteins to make disulfide bonds. Gene fusion approaches were used to show that (i) DsbB is a membrane protein which spans the membrane four times and (ii) both the N- and C-termini of the protein are in the cytoplasm. Mutational analysis shows that of the six cysteines in DsbB, four are necessary for proper DsbB function in vivo. Each of the periplasmic domains of the protein has two essential cysteines. The two cysteines in the first periplasmic domain are in a Cys-X-Y-Cys configuration that is characteristic of the active site of other proteins involved in disulfide bond formation, including DsbA and protein disulfide isomerase.     

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.     

Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3

BACKGROUND: Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped. RESULTS: We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation. CONCLUSIONS: We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.     

Mechanism of integrin activation by disulfide bond reduction

Integrin alphaIIbbeta3 plays a pivotal role in hemostasis and thrombosis by mediating platelet adhesion and platelet aggregation. Integrin alphaIIbbeta3 contains an on/off switch that regulates its ligand binding affinity. The switch from "off" to "on" is commonly referred to as integrin activation. We recently identified a redox site within the extracellular domain of the platelet integrin alphaIIbbeta3 that exhibits many properties that one might expect of the on/off switch [Yan, B., and Smith, J. W. (2000) J. Biol. Chem. 275, 39964-39972]. Several independent reports show that reducing agents, such as dithiothreitol, can activate integrins. The objective of the present study was to determine if the effects of DTT can be attributed to a perturbation at the integrin redox site. Indeed, we find that DTT reduces two disulfide bonds within the integrin's cysteine-rich domain. Such bond reduction leads to global conformational changes within both alphaIIb and beta3 and the opening of the RGD and fibrinogen binding sites. These findings causally link the reduction of disulfide bonds within the integrin's redox site to transitions in the integrin's activation state.     

Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum

In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide bonds in Ero1p. Cys100, Cys105, Cys352, and Cys355 of Ero1p are important for oxidative protein folding and for cell viability, whereas Cys90, Cys208, and Cys349 are dispensable for these functions. Substitution of Cys100 with alanine impedes the capture of Ero1p-Pdi1p mixed-disulfide complexes from yeast, and also blocks oxidation of Pdi1p in vivo. Cys352 and Cys355 are required to maintain the fully oxidized redox state of Ero1p, and also play an auxiliary role in thiol-disulfide exchange with Pdi1p. These results suggest a model for the function of Ero1p wherein Cys100 and Cys105 form a redox-active disulfide bond that engages directly in thiol-disulfide exchange with ER oxidoreductases. The Cys352-Cys355 disulfide could then serve to reoxidize the Cys100-Cys105 cysteine pair, possibly through an intramolecular thiol-disulfide exchange reaction.     

The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit beta1 (Y783 and Y795) to phenylalanines markedly reduces the capability of beta1A integrins to mediate directed cell migration. In this study, beta1-dependent cell spreading was found to be delayed in GD25 cells expressing beta1A(Y783/795F) compared to that in wild-type GD25-beta1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to beta1-dependent adhesion in GD25-beta1A(Y783/795F) cells compared to that in wild-type GD25-beta1A or mutants in which only a single tyrosine was altered (beta1A(Y783F) or beta1A(Y795F)). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via beta1A(Y783/795F) lies at the level of the initial autophosphorylation step. Indeed, beta1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the beta1A(Y783/795F) cells, consistent with the impairment in FAK activation. In contrast, p130(CAS) overall tyrosine phosphorylation was unaffected by the beta1 mutations. Despite the defect in beta1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-beta1A(Y783/795F) cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit beta1A are critical mediators of FAK activation and cell spreading in GD25 cells.     

A single disulfide bond differentiates aggregation pathways of beta2-microglobulin

Deposition of wild-type beta2-microglobulin (beta2m) into amyloid fibrils is a complication in patients undergoing long-term hemodialysis. The native beta-sandwich fold of beta2m has a highly conserved disulfide bond linking Cys25 and Cys80. Oxidized beta2m forms needle-like amyloid fibrils at pH 2.5 in vitro, whereas reduced beta2m, at acid pH, in which the intra-chain disulfide bond is disrupted, cannot form typical fibrils. Instead, reduced beta2m forms thinner and more flexible filaments. To uncover the difference in molecular mechanisms underlying the aggregation of the oxidized and reduced beta2m, we performed molecular dynamics simulations of beta2m oligomerization under oxidized and reduced conditions. We show that, consistent with experimental observations, the oxidized beta2m forms domain-swapped dimer, in which the two proteins exchange their N-terminal segments complementing each other. In contrast, both dimers and trimers, formed by reduced beta2m, are comprised of parallel beta-sheets between monomers and stabilized by the hydrogen bond network along the backbone. The oligomerized monomers are in extended conformations, capable of further aggregation. We find that both reduced and oxidized dimers are thermodynamically less stable than their corresponding monomers, indicating that beta2m oligomerization is not accompanied by the formation of a thermodynamically stable dimer. Our studies suggest that the different aggregation pathways of oxidized and reduced beta2m are dictated by the formation of distinct precursor oligomeric species that are modulated by Cys25-Cys80 disulfide-bonds. We propose that the propagation of domain swapping is the aggregation mechanism for the oxidized beta2m, while "parallel stacking" of partially unfolded beta2m is the aggregation mechanism for the reduced beta2m.     

Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. Beta1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on beta1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and beta1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.     

Protein engineering of a disulfide bond in a beta/alpha-barrel protein.

A disulfide bond has been introduced in the beta/alpha-barrel enzyme N-(5'-phosphoribosyl)anthranilate isomerase from Saccharomyces cerevisiae. The design of this disulfide bond was based on a model structure of this enzyme, built from the high-resolution crystal structure of the N-(5'-phosphoribosyl)anthranilate isomerase domain from Escherichia coli. The disulfide cross-link is spontaneously formed in vitro between residues 27 and 212, located in the structurally adjacent alpha-helices 1 and 8 of the outer helical ring of the beta/alpha-barrel. It creates a loop of 184 residues that account for 83% of the sequence of this enzyme, thus forming a quasi circular protein. The cross-linked mutant enzyme displays wild-type steady-state kinetic parameters. Measurements of the equilibrium constant for the reduction of this disulfide bond by 1,4-dithiothreitol show that its bond strength is comparable to that of other engineered protein disulfide bonds. The oxidized, cross-linked N-(5'-phosphoribosyl)anthranilate isomerase mutant is about 1.0 kcal/mol more stable than the wild-type enzyme, as estimated from its equilibrium unfolding transitions by guanidine hydrochloride.     

Amyloidogenic synthetic peptides of beta2-microglobulin--a role of the disulfide bond

To search for the essential regions responsible for the beta2-microglobulin (beta2-m) amyloid fibril formation, we synthesized six peptides corresponding to six of the seven beta-sheets in the native structure of beta2-m, and examined their amyloidogenicity. Among the peptides examined, peptide (21-31) (strand B) and the mixture of peptide (21-31) and (78-86) (strand F) showed fibril formation at both pH 2.5 and 7.5. Peptide (21-31) is the N-terminal half of the previously reported proteolytic fragment of beta2-m, Ser21-Lys41 (K3), suggesting that this region may be the essential core. Interestingly, the dimer formation of peptide (21-31) by the disulfide bond substantially facilitated the fibril formation, indicating that the disulfide bond is important for the structural stability of the fibrils.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.     

Identification of human vesicle monoamine transporter (VMAT2) lumenal cysteines that form an intramolecular disulfide bond

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. To obtain structural information regarding this large membrane protein by analysis of disulfide bonds and other intramolecular cross-links, we engineered a strategic thrombin cleavage site into deglycosylated, HA-tagged human VMAT2. Insertion of this protease site did not disrupt ligand binding or serotonin transport. Thrombin cleavage at an engineered site in the predicted cytoplasmic loop between transmembrane (TM) domains 6 and 7 (loop 6/7) was rapid and quantitative in the absence of any detergent. The loop 6/7 thrombin site allowed assessment of an intramolecular disulfide bond between the N- and C-terminal halves of the transporter. Consistent with this hypothesis, after quantitative loop 6/7 thrombin cleavage, in the absence of reducing agent, VMAT2 migrated on SDS-polyacrylamide gels as a full-length transporter. Addition of dithiothreitol resulted in complete conversion from full-length to thrombin-cleaved size, demonstrating a DTT-reversible covalent bond. The identity of the disulfide-bound cysteine pair was suggested by the observation that replacement of Cys 126 or Cys 333 with serine both reduced [(3)H]serotonin transport. Replacement of either Cys 126 or Cys 333 was found to eliminate the DTT-reversible intramolecular covalent bond. We conclude that human VMAT2 Cys 126 in loop 1/2 and Cys 333 in loop 7/8 form a disulfide bond which contributes to efficient monoamine transport.     

Evidence for activation of tissue factor by an allosteric disulfide bond

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.     

Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.     

PKR-dependent and -independent mechanisms are involved in translational shutoff during Sindbis virus infection

The replication of Sindbis virus (SIN) profoundly affects the metabolism of infected vertebrate cells. One of the main events during SIN infection is the strong inhibition of translation of cellular mRNAs. In this study, we used a combination of approaches, including the study of SIN replication in PKR(-/-) mouse embryo fibroblasts or in the presence of an excess of catalytically inactive PKR. We show that the PKR-dependent inhibition of translation is not the only and most likely not the major pathway mediating translational shutoff during SIN infection. The PKR-independent mechanism strongly affects the translation of cellular templates, whereas translation of SIN subgenomic RNA is resistant to inhibition, and this leads to a benefit for viral replication. Our findings suggest that both PKR-dependent and non-PKR-dependent mechanisms of SIN-induced translational shutoff can be manipulated by using SIN replicons expressing mutated SIN nsP2 or kinase-defective PKR. Specifically, we show that expression of heterologous genes from SIN-based and most likely other alphavirus-based replicons can be increased by downregulating both the PKR-dependent and PKR-independent translational shutoffs.     

Identification of disulfide bonds among the nine core 2 N-acetylglucosaminyltransferase-M cysteines conserved in the mucin beta6-N-acetylglucosaminyltransferase family

Bovine core 2 beta1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin beta1,6-N-acetylglucosaminides, including core 2, core 4, and blood group I structures. These structures expand the complexity of mucin carbohydrate structure and thus the functional potential of mucins. The four known mucin beta1,6-N-acetylglucosaminyltransferases contain nine conserved cysteines. We determined the disulfide bond assignments of these cysteines in [(35)S]cysteine-labeled bC2GnT-M isolated from the serum-free conditioned medium of Chinese hamster ovary cells stably transfected with a pSecTag plasmid. This plasmid contains bC2GnT-M cDNA devoid of the 5'-sequence coding the cytoplasmic tail and transmembrane domain. The C18 reversed phase high performance liquid chromatographic profile of the tryptic peptides of reduced-alkylated (35)S-labeled C2GnT-M was established using microsequencing. Each cystine pair was identified by rechromatography of the C8 high performance liquid chromatographic radiolabeled tryptic peptides of alkylated bC2GnT-M on C18 column. Among the conserved cysteines in bC2GnT-M, the second (Cys(113)) was a free thiol, whereas the other eight cysteines formed four disulfide bridges, which included the first (Cys(73)) and sixth (Cys(230)), third (Cys(164)) and seventh (Cys(384)), fourth (Cys(185)) and fifth (Cys(212)), and eighth (Cys(393)) and ninth (Cys(425)) cysteine residues. This pattern of disulfide bond formation differs from that of mouse C2GnT-L, which may contribute to the difference in substrate specificity between these two enzymes. Molecular modeling using disulfide bond assignments and the fold recognition/threading method to search the Protein Data Bank found a match with aspartate aminotransferase structure. This structure is different from the two major protein folds proposed for glycosyltransferases.