Winfried Kammer

ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.

Activity patterns of proteasome subunits reflect bortezomib sensitivity of hematologic malignancies and are variable in primary human leukemia cells.

Proteasomal proteolysis relies on the activity of six catalytically active proteasomal subunits (β1, β2, β5, β1i, β2i and β5i). Applying a functional proteomics approach, we used a recently developed activity-based, cell-permeable proteasome-specific probe that for the first time allows differential visualization of individual active proteasomal subunits in intact primary cells. In primary leukemia samples, we observed remarkable variability in the amounts of active β1/1i-, β2/2i- and β5/5i-type of subunits, contrasting with their constant protein expression. Bortezomib inhibited β5- and β1-type, but to a lesser extend β2-type of subunits in live primary cells in vitro and in vivo. When we adapted the bortezomib-sensitive human acute myeloid leukemia cell line HL-60 to bortezomib 40 nM (HL-60a), proteasomal activity profiling revealed an upregulation of active subunits, and residual β1/β5-type of activity could be visualized in the presence of bortezomib 20 nM, in contrast to control cells. In a panel of cell lines from hematologic malignancies, the ratio between β2-type and (β1+β5)-type of active proteasomal polypeptides mirrored different degrees of bortezomib sensitivity. We thus conclude that the proteasomal activity profile varies in primary leukemia cells, and that the pattern of proteasomal subunit activity influences the sensitivity of hematologic malignancies toward bortezomib.

Improved sensitivity proteomics by postharvest alkylation and radioactive labeling of proteins

We describe approaches to improve the detection of proteins by postharvest alkylation and subsequent radioactive labeling with either [3H]iodoacetamide or 125I. Database protein sequence analysis suggested that cysteine is not suitable for detection of the entire proteome, but that cysteine alkylating reagents can increase the number of proteins able to be detected by iodination chemistry. Proteins were alkylated with β‐(4‐hydroxyphenyl)ethyl iodoacetamide, or with 1,5‐l‐AEDANS (the Hudson Weber reagent). Subsequent iodination using the Iodo‐Gen system was found to be most efficient. The enhanced sensitivity obtainable by using these approaches is expected to be sufficient for visualization of the lowest copy number proteins from human cells, such as from clinical samples. However, we argue that significantly improved methods of protein separation will be necessary to resolve the large number of proteins expected to be detectable with this sensitivity.

Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner

Endocytic proteolysis represents a major functional component of the major histocompatibility complex class II antigen‐presentation machinery. Although transport and assembly of class II molecules in the endocytic compartment are well characterized, we lack information about the pattern of endocytic protease activity along this pathway. Here, we used chemical tools that visualize endocytic proteases in an activity‐dependent manner in combination with subcellular fractionation to dissect the subcellular distribution of the major cathepsins (Cat) CatS, CatB, CatH, CatD, CatC, and CatZ as well as the asparagine‐specific endoprotease (AEP) in human B‐lymphoblastoid cells (BLC). Endocytic proteases were distributed in two distinct patterns: CatB and CatZ were most prominent in early and late endosomes but absent from lysosomes, and CatH, CatS, CatD, CatC, and AEP distributed between late endosomes and lysosomes, suggesting that CatB and CatZ might be involved in the initial proteolytic attack on a given antigen. The entire spectrum of protease activity colocalized with human leukocyte antigen‐DM and the C‐terminal and N‐terminal processing of invariant chain (Ii) in late endosomes. CatS was active in all endocytic compartments. Surprisingly and in contrast with results from dendritic cells, inhibition of CatS activity by leucine–homophenylalanine–vinylsulfone‐phenol prevented N‐terminal processing of Ii but did not alter the subcellular trafficking or surface delivery of class II complexes, as deferred from pulse‐chase analysis in combination with subcellular fractionation and biotinylation of cell‐surface protein. Thus, BLC contain distinct activity patterns of proteases in endocytic compartments and regulate the intracellular transport and surface‐delivery of class II in a CatS‐independent manner.

Proteomic Identification of the Involvement of the Mitochondrial Rieske Protein in Epilepsy

Summary:  Purpose: Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague–Dawley rat is associated with a critical developmental interval of 25 ± 1 days; the identification of this long, well‐defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relation to normal and pathologic brain function.

Demonstration of PDC-E1 subunits as major antigens in the complement-fixing fraction M4 and re-evaluation of PDC-E1-specific antibodies in PBC patients.

Abstract: Background: Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen.

Comparative proteomic analysis of neoplastic and non-neoplastic germ cell tissue.

Abstract A comparative proteomic analysis of neoplastic versus non-neoplastic seminoma identified glutathione S-transferase M3 as a differentially expressed protein. This expression difference could also be observed at the mRNA level, implying neoplasm-associated alterations in transcriptional or post-transcriptional mechanisms.