High resolution analysis of proteolytic substrate processing.

Members of the widely conserved HtrA family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, circular dichroism spectroscopy and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other post-translational modifications such as phosphorylation in dynamic protein complexes.

HTRA1-Dependent Cell Cycle Proteomics.

The HTRA1 gene encoding an evolutionary conserved protein quality-control factor can be epigenetically silenced or inactivated by mutation under pathologic conditions such as cancer. Recent evidence suggests that the loss of HTRA1 function causes multiple phenotypes, including the acceleration of cell growth, delayed onset of senescence, centrosome amplification, and polyploidy, suggesting an implication in the regulation of the cell cycle. To address this model, we performed a large-scale proteomics study to correlate the abundance of proteins and HTRA1 levels in various cell cycle phases using label-free-quantification mass spectrometry. These data indicate that the levels of 4723 proteins fluctuated in a cell-cycle-dependent manner, 2872 in a HTRA1-dependent manner, and 1530 in a cell-cycle- and HTRA1-dependent manner. The large number of proteins affected by the modulation of HTRA1 levels supports its general role in protein homeostasis. Moreover, the detected changes in protein abundance, in combination with pull-down data, implicate HTRA1 in various cell cycle events such as DNA replication, chromosome segregation, and cell-cycle-dependent apoptosis. These results highlight the wide implications of HTRA1 in cellular physiology.

Identification of the Natural Product Rotihibin†A as a TOR Kinase Signaling Inhibitor by Unbiased Transcriptional Profiling.

Bioactive natural products are important starting points for developing chemical tools for biological research. For elucidating their bioactivity profile, biological systems with concise complexity such as cell culture systems are frequently used, whereas unbiased investigations in more complex multicellular systems are only rarely explored. Here, we demonstrate with the natural product Rotihibin A and the plant research model system Arabidopsis thaliana that unbiased transcriptional profiling enables a rapid, label-free, and compound economic evaluation of a natural product's bioactivity profile in a complex multicellular organism. To this end, we established a chemical synthesis of Rotihibin A as well as that of structural analogues, followed by transcriptional profiling-guided identification and validation of Rotihibin A as a TOR signaling inhibitor (TOR=target of rapamycin). These findings illustrate that a combined approach of transcriptional profiling and natural product research may represent a technically simple approach to streamline the development of chemical tools from natural products even for biologically complex multicellular biological systems.

Rational correction of pathogenic conformational defects in HTRA1.

Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.

Targeted Protein Unfolding at the Golgi Apparatus.

Maintaining protein homeostasis (proteostasis) is vital to cellular and organismal health. How the Golgi apparatus, the central protein maturation and sorting station in the cell, manages misfolded proteins to maintain proteostasis is still poorly understood. Here we present a strategy for targeted protein unfolding at the Golgi that enables studying Golgi-related protein quality control and stress-signaling pathways. Targeted protein unfolding is induced by small molecule-based chemical biology approaches-hydrophobic tagging and the use of a destabilization domain. Imaging studies allow visualizing quality control (QC) phenotypes, such as the formation of QC carriers and Golgi-to-endoplasmic reticulum trafficking, and correlating these phenotypes with other trafficking processes.

Chemical Validation of DegS As a Target for the Development of Antibiotics with a Novel Mode of Action.

Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first in vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway σE represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.