Summary
The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest. Loss of a ubiquitin‐related system ...(URM1–UBA4 ) and KTI11 enhances post‐translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function. Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p). Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p. Among the pool that co‐fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates. Thus, modification/proteolysis may affect association of Elongator with pol II or its localization. Consistently, an Elongator‐nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency. Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS‐ and karyopherin‐dependent nuclear import.
Toxin-specific genes are often located on mobile genetic elements such as phages, plasmids and pathogenicity islands (PAIs). The uropathogenic
E. coli strain 536 carries two α-hemolysin gene ...clusters, which are part of the pathogenicity islands I
536 and II
536, respectively. Using different genetic techniques, two additional PAIs were identified in the genome of the
E. coli strain 536, and it is likely that further PAIs are located on the genome of this strain. Pathogenicity islands are often associated with tRNA genes. In the case of the
E. coli strain 536, the PAI-associated tRNA gene
leuX, which encodes a minor leucyl-tRNA, affects the expression of various virulence traits including α-hemolysin production. The exact mode of action of the tRNA
5
Leu-dependent gene expression has to be identified in the future.
A recombinant phospholipase D from white cabbage (PLD2) composed of 812 amino acid residues was studied by site-directed mutagenesis and limited proteolysis to obtain first information on its ...tertiary structure. Limited proteolysis by thermolysin resulted in the formation of some large fragments of PLD2. From mass spectrometry and N-terminal sequencing of the peptides, the cleavage sites could be identified (1. Thr41-Ile42, 2. Asn323-Leu324 or Gly287-Leu288 and Ser319-Ile320 in case of the mutant L324S-PLD2). This suggested an exposed loop in the C2 domain of PLD2 and a large flexible region close to the N-terminal side of the first catalytic (HKD) motif. Calcium ions, the substrate 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and the competitive inhibitor 1,3-dipalmitoylglycero-2-phosphocholine influenced the proteolytic cleavage. Calcium ions exerted a destabilizing effect on the conformation of PLD2.
Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to ...containNG,NG-dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli werein vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, withS-adenosyl-l-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.
Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The
cis/
trans isomer specificity underlying ...ERK2-catalyzed phosphate transfer leads to a complete discrimination of the
cis Ser(Thr)-Pro conformer of oligopeptide substrates.
We investigated
in vitro the ERK2-catalyzed phosphorylation of
Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the
cis Ser54-Pro and the
trans Ser72-Pro moiety. Despite
trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency
k
cat/
K
M of 425 M
−1
s
−1 and 1228 M
−1
s
−1, respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl
cis/
trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl
cis/
trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor
trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.
A novel peptidyl-prolyl cis/trans isomerase was isolated from Escherichia coli cell extract and characterized partially. Determination of the molecular mass by electrospray mass spectrometry ...indicated a protein of 10102 +/- 2 Da, smaller than cyclophilins or FK 506 binding proteins currently known. The specificity constant kcat/Km determined with Succinyl-Ala-Xaa-Pro-Phe-4-nitroanilide (Xaa = Leu) had a value comparable to those from cyclophilins from the same organism. However, the pattern of subsite specificity (Xaa = Gly, Ala, Val, Ile, Leu, Phe, Trp, His, Lys and Glu) was reminiscent of FK506 binding peptidyl-prolyl cis/trans isomerases. The enzyme activity was not inhibited by cyclosporin A or FK506 at inhibitor concentrations of < 5 microM, concentrations that affect most bacterial peptidyl-prolyl cis/trans isomerases. Computer-assisted analysis of 21 amino acid residues of the N-terminus determined by Edman degradation revealed no homology to known peptidyl-prolyl cis/trans isomerases.
A novel class of competitive, acylating inhibitors for the proline-specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor ...molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N-terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting
K
1 values in the μM range. Boc-protected (ω-
N-hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching
K, values of 30 nM to 60 μM. Additionally, α-
N-BOC-(ω-
N-hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time-dependent reaction.
Human parvulin 14 (
hPar14) is a folding helper enzyme belonging to the parvulin family of peptidyl-prolyl
cis/
trans isomerases (PPIases). This enzyme is thought to play a role in cell-cycle and ...chromatin remodeling. Although
hPar14 was nuclearly localized and bound to double-stranded DNA, the molecular basis of the subcellular localization and the functional regulation remained unknown.
Here we show that subcellular localization and DNA-binding ability of
hPar14 is regulated by posttranslational modification of its N-terminal domain. As proved by MALDI-TOF mass spectrometry and MS/MS fragmentation,
hPar14 is phosphorylated at Ser19
in vitro and
in vivo. In human HeLa cells the protein is most likely modified by casein kinase 2 (CK2). Phosphorylation of
hPar14 is inhibited both
in vitro and
in vivo by 5,6-dichloro-1-β-
d-ribofuranosyl benzimidazole (DRB), a specific inhibitor of CK2 activity. Mutation of Ser19 to Ala abolishes phosphorylation and alters the subcellular localization of
hPar14 from predominantly nuclear to significantly cytoplasmic. Immunostaining shows that a Glu19 mutant of
hPar14, which mimics the phosphorylated state of Ser19, is localized around the nuclear envelope, but does not penetrate into the nucleoplasm. In contrast to wild-type
hPar14, the
in vitro DNA-binding affinity of the Glu19 mutant is strongly reduced, suggesting that only the dephosphorylated protein is the active DNA-binding form of
hPar14 in the nucleus.
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