Biomedical subjects

Jianzhong Tang

Publications and source records attributed to Jianzhong Tang.

6 recordsLinked to original sources

PubMed2005

Enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures: spatial propagation of differential flexibility in rubredoxin hybrids.

The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in hyperthermophile proteins below room temperature. Substitution of the hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14-33(Pf)) Cp, that exhibits a 12 degrees increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14-33(Pf)) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14-33(Pf)) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus hyperthermophile homologs.

Amides↗

PubMed2005

Contribution of the multi-turn segment in the reversible thermal stability of hyperthermophile rubredoxin: NMR thermal chemical exchange analysis of sequence hybrids.

Pyrococcus furiosus (Pf) rubredoxin is the most thermostable protein characterized to date. Reflecting the complications arising from irreversible denaturation of this protein, predictions of which structural regions confer differential thermal stability have utilized kinetic stability measurements, hydrogen exchange protection factors, long range hydrogen bond NMR spin couplings, and molecular dynamics simulations, and have primarily implicated the three-stranded beta-sheet and the adjacent metal binding site. Herein, NMR chemical exchange experiments demonstrate reversible two-state unfolding at the thermal transition temperature (T(m)) for hybrids of Pf and the mesophile Clostridium pasteurianum (Cp) rubredoxins which interchange residues 14-33, the so-called multi-turn segment. This complementary pair of hybrid rubredoxins exhibits largely additive incremental thermal stabilizations vs. the parental proteins. Both stabilization free energy measurements as well as incremental T(m) values indicate that a minimum of 37% of the total differential thermal stability resides in this multi-turn segment. Such a proportionality between DeltaDeltaG and incremental T(m) values is predicted for hybrid pairs exhibiting thermodynamic additivity in which the differential stability is predominantly enthalpic.

Amino Acid Sequence↗

PubMed2004

Absence of kinetic thermal stabilization in a hyperthermophile rubredoxin indicated by 40 microsecond folding in the presence of irreversible denaturation.

The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability. This in turn implies a corresponding retardation of the folding reaction. A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus (Pf), which exhibits an irreversible denaturation lifetime at 100 degrees C of nearly a week. Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum (Cp) rubredoxin demonstrate reversible thermal transition temperatures of 144 degrees C (137 degrees C for the N-terminal modified A2K variant) and 104 degrees C, respectively, with similar (un)folding rates of approximately 25,000 s(-1), only modestly slower than the diffusion controlled rate. The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability. The two-state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature (T(m)) of nearly 200 degrees C. This discrepancy is resolved by the observation that the base-catalyzed hydrogen exchange of the model dipeptide (N-acetyl-L-cysteine-N-methylamide)4-Cd2+ is 23-fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data.

Amino Acid Substitution↗

PubMed2004

Increased peptide deformylase activity for N-formylmethionine processing of proteins overexpressed in Escherichia coli: application to homogeneous rubredoxin production.

Deformylation of the initiator N-formylmethionine does not always proceed to completion for proteins overexpressed in Escherichia coli. To overcome this limitation, the def gene encoding the Escherichia coli peptide deformylase was cloned into the plysS plasmid under the tetracycline (Tc) promoter control. The efficiency of this constitutive level of peptide deformylase expression was demonstrated for the case of the rubredoxins from both mesophilic and hyperthermophilic organisms which normally retain a majority of their N-formyl terminal form. Indicating the potential structural/functional significance of residual formylation, the presence of a highly solvent exposed N-formyl group in rubredoxin is discernable in the amide NMR chemical shifts for the active site metal-coordinating cysteines more than 21A away.

Amidohydrolases↗

PubMed2002

Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene.

Several proteases and their co-expressed inhibitors modulate the interdependent processes of cell migration and matrix proteolysis during wound repair. Transcription of the gene encoding plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor important in the control of barrier proteolysis and cell-to-matrix adhesion, is spatially-temporally regulated following epithelial denudation injury in vitro as well as in vivo. Using a well-defined culture model of acute epidermal wounding and reepithelialization, PAI-1 mRNA/protein synthesis was induced early after monolayer scraping and restricted to cells comprising the motile cohort. PAI-1 levels in locomoting cells remained elevated (relative to the distal, contact-inhibited monolayer regions) throughout the time course of trauma repair. Targeted PAI-1 downregulation by transfection of antisense PAI-1 expression constructs significantly impaired keratinocyte migration and monolayer scrape wound closure. Injury-induced PAI-1 transcription closely paralleled growth state-dependent controls on the PAI-1 gene. An E-box motif (CACGTG) in the PAI-1 proximal promoter (located at nucleotides -160 to -165), previously shown to be necessary for serum-induced PAI-1 expression, was bound by nuclear factors from wound-stimulated but not quiescent, contact-inhibited, keratinocytes. UV crosslinking approaches to identify E-box-binding factors coupled with deoxyoligonucleotide affinity chromatography and gel retardation assays confirmed at least one major E-box-binding protein in both serum- and wound-activated cells to be USF-1, a member of the helix-loop-helix family of transcription factors. An intact hexanucleotide E-box motif was necessary and sufficient for USF-1 binding using nuclear extracts from both serum- and wound-simulated cells. Two species of immunoreactive USF-1 were identified by western blotting of total cellular lysates that corresponded to the previously characterized phosphorylated and non-phosphorylated forms of the protein. USF-1 isolated by PAI-1 promoter-DNA affinity chromatography was almost exclusively phosphorylated. Only a fraction of the total cellular USF-1 in proliferating cultures, by comparison, was phosphorylated at any given time. PAI-1 E-box binding activity, assessed by probe mobility shift criteria, increased within 2 hours of monolayer scrape injury, a time frame consistent with wound-stimulated increases in PAI-1 transcription. Relative to intact cultures, scrape site-juxtaposed cells had significantly greater cytoplasmic and nuclear USF-1 immunoreactivity correlating with the specific in situ-restricted expression of PAI-1 transcripts/protein in the wound-edge cohort. USF-1 immunocytochemical staining declined significantly with increasing distance from the denudation site. These data are the first to indicate that binding of USF-1 to its target motif can be induced by 'tissue' injury in vitro and implicate USF-1 as a transcriptional regulator of genes (e.g. PAI-1) involved in wound repair.

Animals↗

PubMed2002

Cyclin-dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii.

The Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission. We tested the role of TPK2, a homologue of the CDC2 cyclin-dependent kinases, in cell cycle regulation. TPK2 tagged with HA epitope (TPK2-HA-wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies. TPK2-HA-wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase. TPK2-HA-wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E. Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains. Overexpression of a dominant-negative mutant of TPK2 (TPK2-HA-dn) in T. gondii tachyzoites arrested replication. FACS analysis of tachyzoites expressing TPK2-HA-dn revealed an increase in the fraction of cells in S-phase when compared with TPK2-HA-wt transfected parasites. Expression of TPK2-HA-wt did not arrest tachyzoite replication. No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T. gondii from most other eukaryotic cells. These data suggest that TPK2 executes an essential function in T. gondii cell cycle and is likely to be the T. gondii CDC2 orthologue.

Amino Acid Sequence↗