Proteomic data and structure analysis combined reveal interplay of structural rigidity and flexibility on selectivity of cysteine cathepsins.

Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning.

A protein-protein interaction map of the TNF-induced NF-κB signal transduction pathway.

Tumor Necrosis Factor (TNF) has a crucial role in inflammation, cell proliferation and cell death. Dysregulation of TNF receptor 1 (TNFR1)-induced Nuclear Factor-kappa B (NF-κB) signaling leads to chronic inflammation and is associated with several human inflammatory pathologies. Hence, TNF neutralization suppresses inflammation and attenuates inflammatory pathology.

Identification of Immune-Responsive Gene 1 (IRG1) as a Target of A20.

A20 is a negative regulator of NF-κB signaling; it controls inflammatory responses and ensures tissue homeostasis. A20 is thought to restrict NF-κB activation both by its ubiquitin-editing activity as well as by its nonenzymatic activities. Besides its role in NF-κB signaling, A20 also acts as a protective factor inhibiting apoptosis and necroptosis.

Analyzing trapped protein complexes by Virotrap and SFINX.

The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption of such networks is associated with many pathologies. Virtually all the approaches to the study of protein complexes require cell lysis, a dramatic step that obliterates cellular integrity and profoundly affects protein interactions.

Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry.

Protein complexes are essential in all organizational and functional aspects of the cell. Different strategies currently exist for analyzing such protein complexes by mass spectrometry, including affinity purification (AP-MS) and proximal labeling-based strategies. However, the high sensitivity of current mass spectrometers typically results in extensive protein lists mainly consisting of nonspecifically copurified proteins.

Trapping mammalian protein complexes in viral particles.

Cell lysis is an inevitable step in classical mass spectrometry-based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification.

Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S.

Proteases are important effectors of numerous physiological and pathological processes. Reliable determination of a protease's specificity is crucial to understand protease function and to develop activity-based probes and inhibitors. During the last decade, various proteomic approaches for profiling protease substrate specificities were reported.