Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups flanking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings.     

Geochemical and physiological evidence for mixed aerobic and anaerobic field biodegradation of coal tar waste by subsurface microbial communities

We used geochemical analyses of groundwater and laboratory-incubated microcosms to investigate the physiological responses of naturally occurring microorganisms to coal-tar-waste constituents in a contaminated aquifer. Waters were sampled from wells along a natural hydrologic gradient extending from uncontaminated (1 well) into contaminated (3 wells) zones. Groundwater analyses determined the concentrations of carbon and energy sources (pollutants or total organic carbon), final electron acceptors (oxygen, nitrate, sulfate), and metabolic byproducts (dissolved inorganic carbon [DIC], alkalinity, methane, ferrous iron, sulfide, Mn2+). In the contaminated zone of the study site, concentrations of methane, hydrogen, alkalinity, and DIC were enhanced, while dissolved oxygen and nitrate were depleted. Field-initiated biodegradation assays using headspace-free serum bottle microcosms filled with groundwater examined metabolism of the ambient organic contaminants (naphthalene, 2-methylnaphthalene, benzothiophene, and indene) by the native microbial communities. Unamended microcosms from the contaminated zone demonstrated the simultaneous degradation of several coal-tar-waste constituents at the in situ temperature (10°C). Lag phases prior to the onset of biodegradation indicated the prevalence of both aerobic and anaerobic conditions in situ. Electron acceptor-amended microcosms from the most contaminated well waters demonstrated only aerobic naphthalene degradation. Collectively, the geochemical and microbial evidence show that biodegradation of coal-tar-waste constituents occurs via both aerobic and anaerobic terminal electron accepting processes at this site.     

Stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post‐translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells. Pharmacologic Pim‐1 inhibition using the small molecule inhibitor CX‐6258 decreased steady state levels and half‐life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S‐proteasome, demonstrating that Pim‐1 protects NKX3.1 from proteasome‐mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim‐1 phospho‐acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N‐terminal PEST domain is essential for Pim‐1‐mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim‐1. Pim‐1‐mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells. J. Cell. Biochem. 114: 1050–1057, 2013. © 2012 Wiley Periodicals, Inc.     

Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells.

Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells. The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long. The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups. The 3' and 5' end groups of the products were analyzed. Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG. The 5' end group was either dG or dC with equal frequency. Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends. The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity. If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated.     

Characterization of DNA binding activities of over-expressedKpnI restriction endonuclease and modification methylase

The genes encoding theKpnI restriction endonuclease and methyltransferase fromKlebsiella pneumoniae have been cloned and expressed inEscherchia coli using a two plasmid strategy. The gene forKpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone ofKpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression ofKpnI endonuclease to about 15–30% of cellular protein. Both the enzymes were purified using a single Chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes ofKpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.     

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences

The solid‐state conformations of two αγ hybrid peptides Boc‐[Aib‐γ⁴(R)Ile]₄‐OMe 1 and Boc‐[Aib‐γ⁴(R)Ile]₅‐OMe 2 are described. Peptides 1 and 2 adopt C₁₂‐helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C₁₂ motifs. The structure of peptide 2 reveals the formation of eight successive C₁₂ hydrogen‐bonded turns. Average backbone dihedral angles for αγ C₁₂ helices are peptide 1 , Aib; φ (°) = ?57.2 ± 0.8, ψ (°) = ?44.5 ± 4.7; γ⁴(R)Ile; φ (°) = ?127.3 ± 7.3, θ₁ (°) = 58.5 ± 12.1, θ₂ (°) = 67.6 ± 10.1, ψ (°) = ?126.2 ± 16.1; peptide 2 , Aib; φ (°) = ?58.8 ± 5.1, ψ (°) = ?40.3 ± 5.5; ψ⁴(R)Ile; φ (°) = ?123.9 ± 2.7, θ₁ (°) = 53.3 θ 4.9, θ ₂ (°) = 61.2 ± 1.6, ψ (°) = ?121.8 ± 5.1. The tendency of γ⁴‐substituted residues to adopt gauche–gauche conformations about the C^α–C^β and C^β–C^γ bonds facilitates helical folding. The αγ C₁₂ helix is a backbone expanded analog of α peptide 3₁₀ helix. The hydrogen bond parameters for α peptide 3₁₀ and α‐helices are compared with those for αγ hybrid C₁₂ helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.     

Solution conformation of a tetradecapeptide stabilized by two di‐n‐propyl glycine residues

The solution conformation of a designed tetradecapeptide Boc‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐OMe (Dpg‐14) containing two di‐n‐propyl glycine (Dpg) residues has been investigated by ¹H NMR and circular dichroism in organic solvents. The peptide aggregates formed at a concentration of 3 mM in the apolar solvent CDCl₃ were broken by the addition of 12% v/v of the more polar solvent DMSO‐d₆. Successive N_iH N_i+1H NOEs observed over the entire length of the sequence in this solvent mixture together with the observation of several characteristic medium‐range NOEs support a major population of continuous helical conformations for Dpg‐14. Majority of the observed coupling constants ( ) also support ? values in the helical conformation. Circular dichroism spectra recorded in methanol and propan‐2‐ol give further support in favor of helical conformation for Dpg‐14 and the stability of the helix at higher temperature. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.