Free energy calculations on the stability of the 14-3-3ζ protein

Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins.     

Mutations in the 5’ NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity

The 5’ non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5’ NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5’ NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.     

Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.     

Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNA^Phe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNA^Phe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.     

Limiting CDR-H3 diversity abrogates the antibody response to the bacterial polysaccharide α 1→3 dextran

Anti-polysaccharide Ab responses in mice are often oligoclonal, and the mechanisms involved in Ag-specific clone production and selection remain poorly understood. We evaluated the relative contribution of D(H) germline content versus N nucleotide addition in a classic oligoclonal, T-independent Ab response (α 1→3 dextran [DEX]) by challenging adult TdT-sufficient (TdT(+/+)) and TdT-deficient (TdT(-/-)) gene-targeted mice, limited to the use of a single D(H) gene segment (D-limited mice), with Enterobacter cloacae. D-limited mice achieved anti-DEX-specific levels of Abs that were broadly comparable to those of wild-type (WT) BALB/c mice. Sequence analysis of the third CDR of the H chain intervals obtained by PCR amplification of V(H) domain DNA from DEX-specific plasmablasts revealed the near universal presence of an aspartic acid residue (D99) at the V-D junction, irrespective of the composition of the D(H) locus. Although WT mice were able to use germline D(H) (DQ52, DSP, or DST) gene segment sequence, TdT activity, or both to produce D99, all three D-limited mouse strains relied exclusively on N addition. Additionally, in the absence of TdT, D-limited mice failed to produce a DEX response. Coupled with previous studies demonstrating a reduced response to DEX in TdT(-/-) mice with a WT D(H) locus, we concluded that in the case of the anti-DEX repertoire, which uses a short third CDR of the H chain, the anti-DEX response relies more intensely on sequences created by postnatal N nucleotide addition than on the germline sequence of the D(H).     

The down-regulated expression of 14-3-3σ may activate the other six 14-3-3 isoforms and they may take over the role of 14-3-3σ in the tumor genesis

Seven isoforms of 14-3-3 protein family have different functions in the cancer genesis and progress. It is found that six isoforms were up-regulated expression and inclined to sustain the cancer survival. Conversely, 14-3-3σ strongly promotes cancer apoptosis. Its down-regulated expression was found in many cancer tissues and thought to be an early event in the tumor genesis. Interestingly, no suggestions are made about the possible effect that the down-regulated expression of 14-3-3σ activated the other six 14-3-3 isoforms and they take over the role of 14-3-3σ in the tumor genesis. The inactivation of 14-3-3σ in the early stage of tumor genesis is a clue to trigger the other six 14-3-3 isoforms activation.     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

Catalytic roles of the AMP at the 3′ end of tRNAs

Recent reports suggest that the ribosome retains considerable peptidyl transferase activity even when much of the protein of the ribosome is removed and further suggests that rRNA may be the peptidyl transferase. The work here suggests that the AMP residue at the 3 terminus of each tRNA has some catalytic activity both in the esterification reaction and in forming a pseudopeptide, AcGly, and further suggests that whatever peptidyl transferase is, it finds a cooperative substrate in the aminoacyl-AMP at the 3 terminus of tRNA.     

Arrangement of the Template 3′ of the A-Site Codon on the Human 80S Ribosome

The arrangement of the template sequence 3′ of the A-site codon on the 80S ribosome was studied using mRNA analogs containing Phe codon UUU at the 5′ end and a photoreactive perfluoroarylazido group linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which directed the UUU codon to the P site, bringing a modified nucleotide to position +9 or +12 relative to the first nucleotide of the P-site codon. Upon mild UV irradiation of ribosome complexes, the analogs of both types crosslinked to the 18S rRNA and proteins of the 40S subunit. Comparisons were made with the crosslinking patterns of complexes in which an mRNA analog contained a modified nucleotide in position +7 (the crosslinking to 18S rRNA in such complexes has been studied previously). The efficiency of crosslinking to ribosomal components depended on the nature of the modified nucleotide of an mRNA analog and its position on the ribosome. The extent of crosslinking to the 18S rRNA drastically decreased as the modified nucleotide was transferred from position +7 to position +12. The 18S rRNA nucleotides involved in crosslinking were identified. A modified nucleotide in position +9 crosslinked to the invariant dinucleotide A1824/A1825 and variable A1823 in the 3′ minidomain of the 18S rRNA and to S15. The same ribosomal components have earlier been shown to crosslink to modified nucleotides in positions +4 to +7. In addition, all mRNA analogs crosslinked to invariant C1698 in the 3′ minidomain and to conserved region 605–620, which closes helix 18 in the 5′ domain.     

14-3-3ε marks the amyloid-stimulated microglia long-term activation

Microglia-mediated inflammation in the central nervous system is a hallmark of the pathogenesis of several neurodegenerative diseases including Alzheimer's disease. Microglial cells activation follows the deposition of amyloid β fibrils and it is generally considered a triggering factor in the early steps of the onset of Alzheimer's disease. Although the initial engagement of microglia seems to play a neuroprotective role, many lines of evidence indicate that a persistent activation with the production of proinflammatory molecules contributes to dismantle neuronal activity and to induce neuronal loss occurring in neurodegenerative diseases. To date, limited proteomic data are available on activated microglial cells in response to extracellular amyloidogenic peptides. In this study, murine microglial cells have been employed to investigate the effects of amyloid β peptides in triggering microglial activation. The response was monitored at the proteome level through a two-dimensional gel electrophoresis-based approach. Results show only a limited number of differentially expressed proteins, among these a more acidic species of the cytosolic actin, and the 14-3-3ε protein, found significantly upregulated in Aβ-activated cells. 14-3-3ε belongs to a regulatory protein family involved in important cellular processes, including those leading to neurodegenerative diseases, and thus its increased expression suggests a role of this protein in tuning microglia activation.     

Is the “3 by 5” Initiative the Best Approach to Tackling the HIV Pandemic?

BACKGROUND TO THE DEBATE: The World Health Organization (WHO) and its partners aim to treat 3 million people infected with HIV in poor and middle income countries with antiretroviral treatment by the end of 2005. The ambitious "3 by 5" initiative has had its supporters and its critics since its announcement in 2002.     

Basis of the intrinsic flexibility of the Cε3 domain of IgE

Allergic reactions are triggered by the interaction between IgE and its high-affinity receptor, FcεRI. Various studies have mapped the interaction surface between IgE and its cellular receptors to the third constant domain of IgE (Cε3). The isolated Cε3 domain has been shown to exist as a molten globule, and the domain retains significant flexibility within the context of the IgE protein. Here we have analyzed the structural basis of the intrinsic flexibility of this domain. We have compared the sequence of the Cε3 domain to the sequences of other members of the C1 subset of the immunoglobulin superfamily and observed that Cε3 has an unusually high electrostatic charge and an unusually low content of hydrophobic residues. Mutations restoring Cε3 to a more canonical sequence were introduced in an attempt to derive a more structured domain, and several mutants display decreased levels of disorder. Engineered domains of Cε3 with a range of structural rigidities could serve as important tools for the elucidation of the role of flexibility of the Cε3 domain in IgE's biological functions.     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.     

Modeling the interaction of fipronil-related non-competitive antagonists with the GABA β3-receptor

A three-dimensional model of the beta3-homopentamer of the gamma-aminobutyric acid (GABA) receptor/chloride ionophore complex was developed by homology modeling using the cyro-electron microscopy structure of nicotinic acetylcholine as a template. Interactions between the beta3-homopentamer and two classes of fipronil-related non-competitive antagonists were investigated using docking studies. The phenyl groups of these compounds were stabilized by strong hydrophobic and hydrophilic interactions with the rings formed by Thr256 and Ala252. Leu253 and Ile255 were involved mainly in hydrophobic contact with the pyrazole moiety. Different substitution at positions 15, 16 and 17 of the pyrazole ring of fipronil resulted in weakening of the hydrogen bonds and hydrophobic interactions between the beta3-receptor and fipronil-related heterocyclic compounds, which maybe the principal cause of the decreased affinities reported in vitro. Moreover, a good correlation between total binding energies calculated by AutoDock and experimentally determined IC(50) values proved our models to be reasonable in predicting the interaction mode of the antagonist with the GABA beta3-receptor.     

2,3-Dideoxy-3-Phthalimidopentoses in the Synthesis of 3′-Amino-2′,3′-Dideoxynucleosides

Abstract

3′-Amino-3′deoxythymidine is a very effective drug in vivo against L 1210 leukemia. It mives 1441 increase in lifespan with very little drug-induced toricitylil. Therefore, it was attractive to synthesize a large series of cuialogues, but unfortunately, such compounds are only achievable through a 1inear synthesis via the corresponding nucleoside which typically is transformed into the 3′-azido derivative and finally reduced.     

Evidence against H+−HCO 3 − symport as the mechanism for HCO 3 − transport in the cyanobacteriumAnabaena variabilis

Summary The rate of inorganic carbon uptake and its steadystate accumulation ratio (intracellular/extracellular concentration) was determined in the cyanobacteriumAnabaena variabilis as a function of extracellular pH. The free energy of protons ( ) across the plasmalemma was calculated from determinations of membrane potential, and intracellular pH, as a function of the extracellular pH. While inward proton motive force decreased with increasing extracellular pH from 6.5 to 9.5, rate of HCO ₃ ^– influx and its accumulation ration increased. The latter is several times larger than would be expected should HCO ₃ ^– influx be driven by . It is concluded that HCO ₃ ^– transport in cyanobacteria is not driven by the proton motive force.