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Publications - Cell and Metabolic Biology
Publications
Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. Effector protein delivery is controlled by the T3S chaperone HpaB, which presumably escorts effector proteins to the secretion apparatus. One intensively studied effector is the transcription activator-like (TAL) effector AvrBs3, which binds to promoter sequences of plant target genes and activates plant gene expression. It was previously reported that type III-dependent delivery of AvrBs3 depends on the N-terminal protein region. The signals that control T3S and translocation of AvrBs3, however, have not yet been characterized. In the present study, we show that T3S and translocation of AvrBs3 depend on the N-terminal 10 and 50 amino acids, respectively. Furthermore, we provide experimental evidence that additional signals in the N-terminal 30 amino acids and the region between amino acids 64 and 152 promote translocation of AvrBs3 in the absence of HpaB. Unexpectedly, in vivo translocation assays revealed that AvrBs3 is delivered into plant cells even in the absence of HrpF, which is the predicted channel-forming component of the T3S translocon in the plant plasma membrane. The presence of HpaB- and HrpF-independent transport routes suggests that the delivery of AvrBs3 is initiated during early stages of the infection process, presumably before the activation of HpaB or the insertion of the translocon into the plant plasma membrane.
Publications
Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)–electrospray ionization–mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC–MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated.
Publications
Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys549 (KAm(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.
This page was last modified on 27 Jan 2025 .
