Biomedical subjects

Beate Rockel

Publications and source records attributed to Beate Rockel.

9 recordsLinked to original sources

PubMed2006

Size matters for the tripeptidylpeptidase II complex from Drosophila: The 6-MDa spindle form stabilizes the activated state.

Tripeptidylpeptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases, a key component of the protein degradation cascade in many eukaryotes, which cleaves tripeptides from the N terminus of proteasome-released products. The Drosophila TPP II is a large homooligomeric complex (approximately 6 MDa) that is organized in a unique repetitive structure with two strands each composed of ten stacked homodimers; two strands intertwine to form a spindle-shaped structure. We report a novel procedure of preparing an active, structurally homogeneous TPP II holo-complex overexpressed in Escherichia coli. Assembly studies revealed that the specific activity of TPP II increases with oligomer size, which in turn is strongly concentration-dependent. At a TPP II concentration such as prevailing in Drosophila, equilibration of size and activity proceeds on a time scale of hours and leads to spindle formation at a TPP II concentration of > or =0.03 mg/ml. Before equilibrium is reached, activation lags behind assembly, suggesting that activation occurs in a two-step process consisting of (i) assembly and (ii) a subsequent conformational change leading to a switch from basal to full activity. We propose a model consistent with the hyperbolic increase of activity with oligomer size. Spindle formation by strand pairing causes both significant thermodynamic and kinetic stabilization. The strands inherently heterogeneous in length are thus locked into a discrete oligomeric state. Our data indicate that the unique spindle form of the holo-complex represents an assembly motif stabilizing a highly active state.

Aminopeptidases↗

PubMed2006

Characterization of AMA, a new AAA protein from Archaeoglobus and methanogenic archaea.

We have previously reported a new group of AAA proteins, which is only found in Archaeoglobus and methanogenic archaea (AMA). The proteins are phylogenetically basal to the metalloprotease clade and their N-terminal domain is homologous to the beta-clam part of the N-domain of CDC48-like proteins. Here we report the biochemical and biophysical characterization of Archaeoglobus fulgidus AMA, and of its isolated N-terminal (AMA-N) and ATPase (AMA-DeltaN) domains. AfAMA forms hexameric complexes, as does AMA-N, while AMA-DeltaN only forms dimers. The ability to hexamerize is dependent on the integrity of a GYPL motif in AMA-N, which resembles the pore motif of FtsH and HslU. While the physiological function of AMA is unknown, we show that it has ATP-dependent chaperone activity and can prevent the thermal aggregation of proteins in vitro. The ability to interact with non-native proteins resides in the N-domain and is energy-independent.

Adenosine Triphosphatases↗

PubMed2006

20S proteasomes have the potential to keep substrates in store for continual degradation.

The 20S core of the proteasome, which together with the regulatory particle plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically, the role of the antechambers is not fully understood, and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we have shown that by applying both electron microscopy and tandem mass spectrometry (MS) approaches to this multisubunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allows us to conclude that a maximum of three green fluorescent protein and four cytochrome c substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover, we deduce that one green fluorescent protein or two cytochrome c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases where translocation rates are slower than proteolysis. More generally, the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.

Cytochromes c↗

PubMed2005

Particle picking by segmentation: a comparative study with SPIDER-based manual particle picking.

Boxing hundreds of thousands of particles in low-dose electron micrographs is one of the major bottle-necks in advancing toward achieving atomic resolution reconstructions of biological macromolecules. We have shown that a combination of pre-processing operations and segmentation can be used as an effective, automatic tool for identifying and boxing single-particle images. This paper provides a brief description of how this method has been applied to a large data set of micrographs of ice-embedded ribosomes, including a comparative analysis of the efficiency of the method. Some results on processing micrographs of tripeptidyl peptidase II particles are also shown. In both cases, we have achieved our goal of selecting at least 80% of the particles that an expert would select with less than 10% false positives.

Algorithms↗

PubMed2005

VAT, the thermoplasma homolog of mammalian p97/VCP, is an N domain-regulated protein unfoldase.

The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg2+ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VATDeltaN complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.

Adenosine Triphosphatases↗

PubMed2005

Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II.

In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.

Aminopeptidases↗

PubMed2003

Lignin distribution in wood cell walls determined by TEM and backscattered SEM techniques.

The lignin distribution in cell walls of spruce and beech wood was determined by high-voltage transmission-electron-microscopy (TEM) in sections stained with potassium permanganate as well as by field-emission-scanning-electron-microscopy (FE-SEM) combined with a back-scattered electron detector on mercurized specimens. The latter is a new technique based on the mercurization of lignin and the concomitant visualization of mercury by back-scattered electron microscopy (BSE). Due to this combination it was possible to obtain a visualized overview of the lignin distribution across the different layers of the cell wall. To our knowledge, this combined method was used the first time to analyse the lignin distribution in cell walls. In agreement with previous work the highest lignin levels were found in the compound middle lamella and the cell corners. Back-scattered FE-SEM allows the lignin distribution in the pit membrane of bordered pits as well as in the various cell wall layers to be shown. In addition, by using TEM as well as SEM we observed that lignin closely follows the cellulose microfibril orientation in the secondary cell wall. From these observations, we conclude that the polymerisation of monolignols is affected by the arrangement of the polysaccharides which constitute the cell wall.

Cell Wall↗

PubMed2002

A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy.

Tripeptidyl peptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases that is thought to act downstream of the proteasome in the ubiquitin-proteasome pathway. Recently, a key role in a pathway parallel to the ubiquitin-proteasome pathway has been ascribed to TPP II, which forms a giant protease complex in mammalian cells. Here, we report the 900-fold purification of TPP II from Drosophila eggs and demonstrate via cryo-electron microscopy that TPP II from Drosophila melanogaster also forms a giant protease complex. The presented three-dimensional reconstruction of the 57 x 27 nm TPP II complex at 3.3 nm resolution reveals that the 150 kDa subunits form a superstructure composed of two segmented and twisted strands. Each strand is 12.5 nm in width and composed of 11 segments that enclose a central channel.

Aminopeptidases↗

PubMed2002

Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum.

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.

Adenosine Triphosphatases↗