Coordinate regulation of RARgamma2, TBP, and TAF(II)135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells

BACKGROUND: Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes. RESULTS: We show that the RNA polymerase II transcription factor TFIID subunits TBP and TAF(II)135 are selectively depleted in extracts from differentiated F9 cells. In contrast, TBP and TAF(II)135 are readily detected in extracts from differentiated F9 cells treated with proteasome inhibitors showing that their disappearance is due to targeted proteolysis. This regulatory pathway is not limited to F9 cells as it is also seen when C2C12 myoblasts differentiate into myotubes. Targeting of TBP and TAF(II)135 for proteolysis in F9 cells takes place coordinately with that previously reported for the RARγ2 receptor and is delayed or does not take place in RAR mutant F9 cells where differentiation is known to be impaired or abolished. Moreover, ectopic expression of TAF(II)135 delays proteolysis of the RARγ2 receptor and impairs primitive endoderm differentiation at an early stage as evidenced by cell morphology, induction of marker genes and apoptotic response. In addition, enhanced TAF(II)135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant. CONCLUSIONS: These observations indicate that appropriately timed proteolysis of TBP and TAF(II)135 is required for normal F9 cell differentiation. Hence, in addition to transactivators, targeted proteolysis of basal transcription factors also plays an important role in gene regulation in response to physiological stimuli

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063

The 1.8\u2005\uc5 resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using \u3b2-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography

ETUDE DE LA FONCTION ET DE LA REGULATION DE TAFII135 UNE SOUS-UNITE DU TFIID DANS LES CELLULES MURINES

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Slednice, soslednice, padnice, preseki in prebodi splošne ravnine v večpoglednih projekcijah

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2(Bp), a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2(Bp) belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2(Bp) (N-PilO2(Bp)), here reported, is the first structural representative of the PF06864 family. N-PilO2(Bp) presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery

Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease.

There is an urgent need for an effective vaccine against human disease caused by Burkholderia pseudomallei, and although a wide range of candidates have been tested in mice none provide high level protection. We considered this might reflect the inability of these vaccine candidates to protect against chronic disease. Using Q-RT PCR we have identified 6 genes which are expressed in bacteria colonising spleens and lungs of chronically infected mice. Three of the genes (BPSL1897, BPSL3369 and BPSL2287) have been expressed in Escherichia coli and the encoded proteins purified. We have also included BPSL2765, a protein known to induce immune responses associated with a reduced incidence of chronic/recurrent disease in humans. Immunisation of mice with a combination of these antigens resulted in the induction of antibody responses against all of the proteins. Compared with mice immunised with capsular polysaccharide or LolC protein, mice immunised with the combination of chronic stage antigens showed enhanced protection against experimental disease in mice

Redefining the PF06864 Pfam Family Based on Burkholderia pseudomallei PilO2Bp S-SAD Crystal Structure

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2Bp, a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2Bp belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2Bp (N-PilO2Bp), here reported, is the first structural representative of the PF06864 family. N-PilO2Bp presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery

N-PilO2_Bp protein.

<p><b>A.</b> Overall fold of N-PilO2_Bp composed of two α/β topology subdomains, each displaying a mixed β-sheet, separated by a (central) cleft. The bound phosphate ion is shown as spheres. <b>B.</b> Topology diagram of N-PilO2_Bp. This diagram was generated using PDBSum server (<a href="http://www.ebi.ac.uk/pdbsum/" target="_blank">www.ebi.ac.uk/pdbsum/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094981#pone.0094981-Laskowski2" target="_blank">[52]</a>. <b>C</b>. Crystal packing of the phosphate-containing N-PilO2_Bp structure, showing the three crystal packing dimers formed by alternative interactions between four symmetry-related monomers (green, blue, magenta and black). The three interfaces are highlighted by black, blue and red shading. The first ‘dimer’, is formed by the interaction between the green (or blue) and the magenta (or black) monomers and the light green (or light blue) phosphate. The second crystallographic dimer occurs between the magenta and black monomers. The third dimer is formed by the green and blue monomers. <b>D</b>. Stereo view of the electron density map for the residues building the phosphate ion binding pocket. The phosphate ion is shown as sphere; the electron density is contoured at 1.5 sigma level. <b>E.</b> Front and back view of N-PilO2_Bp electrostatic surface potential. The electrostatic potential was calculated using the CCP4MG viewer. Negative (red) and positive (blue) charges, and uncharged (white) surfaces are shown. <b>F.</b> Superposition of the 3D structures of N-PilO2_Bp (cyan; PDB codes 4BYZ and 4BZ0) and N-BfpC (chocolate; PDB code 3VHJ).</p

Crystallographic data-collection statistics.

a<p>Data completeness treats Bijvoët mates independently.</p>b<p>Statistics for the highest resolution shells are given in parentheses.</p>c<p><i>R</i>_merge = ∑<i>_hkl</i>∑<i>_i</i>|<i>I(hkl)_I</i> − < <i>I(hkl)</i> >|/∑<i>_hkl</i>∑<i>_i</i>< <i>I(hkl)_i</i> >.</p>d<p>Substructure determination parameters are from ShelxD.</p>e<p>CC  =  [∑<i>wE_oE</i>_c∑<i>w</i> - ∑<i>wE_o</i>∑<i>wE_c</i>]/{[∑<i>w</i>E_o²∑<i>w -</i> (∑<i>w</i>E_o)²] [∑<i>w</i>E_c²∑<i>w</i> -(∑<i>w</i>E_c)²]}^1/2,</p><p>where <i>w</i> is weight. CC_all/CC_weak is the correlation coefficient for all and weak reflections of the best solution.</p>f<p>FOM, figure of merit  =  | <i>F</i>(<i>hkl</i>)best|/|<i>F</i>(<i>hkl</i>)|; <b>F</b>(<i>hkl</i>)best  =  ∑<i>P</i>(α)<b>F</b>_hkl(α)/∑<i>P</i>(α).</p

Comparison of Tfpb machinery R64 thin pilus variant encoding operons for different microorganisms.

<p>The alignment was performed using tblastx from the Blast suite, and visualized in Artemis Comparison Tool. Conserved protein regions are paired by color-shaded regions; the blue and red colors represent the reverse and forward matches, respectively, and color intensity is proportional to the sequence homology. Genes are represented by arrows; the same arrow color indicates putative orthologs. The grey arrows represent genes lacking homologs among represented <i>pil</i> clusters. The <i>pil</i> cluster sequences were retrieved from GenBank: <i>Tfp7</i> locus from <i>B. pseudomallei</i> (<i>Bp</i>) chromosome 2 complete sequence, BX571966.1; PAPI-1 <i>pil</i> gene cluster from <i>P. aeruginosa</i> (<i>Pa</i>) PA14, AY273869.1; R64 transfer region, AB027308.1; and <i>pil</i> operon from <i>Salmonella enterica</i> (<i>Se</i>) subsp. <i>enterica</i> serovar Paratyphi C strain CN13/87, AY249242.1.</p