If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow—and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis -spliced peptides, whereas trans -spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation.

A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to the development of human diseases. Furthermore, detailed mechanistic interrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however, chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes.

Dissecting the degradation of the Alzheimer’s disease–related protein Tau by the 20 S proteasome at single-residue resolution.

The growth of diffraction-quality crystals and experimental phasing remain two of the main bottlenecks in protein crystallography. Here, the high-affinity copper(II)-binding tripeptide GHK was fused to the N-terminus of a GFP variant and an MBP-FG peptide fusion. The GHK tag promoted crystallization, with various residues (His, Asp, His/Pro) from symmetry molecules completing the copper(II) square-pyramidal coordination sphere. Rapid structure determination by copper SAD phasing could be achieved, even at a very low Bijvoet ratio or after significant radiation damage. When collecting highly redundant data at a wavelength close to the copper absorption edge, residual S-atom positions could also be located in log-likelihood-gradient maps and used to improve the phases. The GHK copper SAD method provides a convenient way of both crystallizing and phasing macromolecular structures, and will complement the current trend towards native sulfur SAD and MR-SAD phasing.

The proteasome degrade most of the proteins in eukaryotic cells, thereby controlling the keycellular processes [...]

The proteasome holoenzyme is the major non-lysosomal protease; its proteolytic activity is essential for cellular homeostasis. Thus, it is an attractive target for the development of chemotherapeutics. While the structural basis of core particle (CP) inhibitors is largely understood, their structural impact on the proteasome holoenzyme remains entirely elusive. Here, we determined the structure of the 26S proteasome with and without the inhibitor Oprozomib. Drug binding modifies the energy landscape of conformational motion in the proteasome regulatory particle (RP). Structurally, the energy barrier created by Oprozomib triggers a long-range allosteric regulation, resulting in the stabilization of a non-productive state. Thereby, the chemical drug-binding signal is converted, propagated and amplified into structural changes over a distance of more than 150 Å from the proteolytic site to the ubiquitin receptor Rpn10. The direct visualization of changes in conformational dynamics upon drug binding allows new ways to screen and develop future allosteric proteasome inhibitors.

The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20 S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20 S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics.

The ubiquitin proteasome pathway is one of the major protein degradation pathways in the cell. This pathway is essential for protein quality control and the regulation of the cell cycle. Proteins subjected to the ubiquitin pathway are cleaved into short peptides by the 26S proteasome. The 26S proteasome consists of the 19S regulatory particle and the 20S core proteasome. Ubiquitinated substrates are recognized through their ubiquitin moiety by the 19S regulatory particle, unfolded and translocated into the 20S core proteasome. Within the 20S proteasome hydrolysis of the protein peptide bonds takes place. The aforementioned significance of proteasome for cell function and the associated fact that the proteasome is a drug target for the treatment of cancer makes it an attractive object for structural analyses. A detailed understanding of the proteasome structure is essential to dissect the function and its modulation by proteins and small molecule effectors. In this thesis the sample quality of purified human 20S proteasomes was improved by biochemical optimization and by the introduction of a chromatography free purification protocol. The newly developed purification protocol was shown to be transferable to the purification of human 26S proteasomes, 20S proteasomes from Saccharomyces cerevisiae, Drosophila melanogaster and Thermoplasma acidophilum. Crystals were grown and structures were solved for all purified 20S proteasomes. The established post-crystallization treatment resulted in human and fruit fly 20S proteasomes crystals diffracting to 2.0 Å on average. The best resolution of 1.8 Å was collected for the human 20S proteasome. The robust purification and crystallization protocol leading to unprecedented resolutions obtained for the human 20S proteasome enabled to reanalyze inhibition mechanism of known 20S proteasome inhibitors. Furthermore, screening efforts using small molecules revealed the diazomethylketone C-3455 as new modulator of the 20S proteasome. Most of the previously described reaction mechanisms could be validated. The largest discrepancy to published reaction mechanisms was found for epoxyketone inhibitors. Instead of the proposed formation of a 1,4-morpholine ring, the newly solved structures suggest the formation of a 1,4-oxazepane ring. The observed reaction mechanism was validated by structural analyses, electronic structure calculations of the reaction pathways and kinetic analyses. Based on these results a new reaction mechanism was proposed for epoxyketone inhibition enabling the design of new 20S proteasome inhibitors. Electron microscopy studies of the 26S proteasome and X-ray crystallographic studies of the fruit fly 20S proteasome revealed for the first time allosteric pathways of the pro- teasome. Inhibition of the proteolytic active sites in the 26S holocomplex influences the dynamics of the 19S lid movements. For the fruit fly 20S proteasome a structural rearrangement in the β5-subunit was observed upon inhibition of the chymotryptic-like site or upon binding of a short peptide activator to the α-rings. In summary, the newly established purification protocol led to 20S/26S proteasome sam- ples of superior quality. This made it possible to analyze structural features of the pro- teasome at resolutions not described before. The established high throughput pipeline for crystallographic analyses of human 20S proteasomes can be used for screening small molecules in future.