Simple, scalable, and ultrasensitive tip-based identification of protease substrates.

Proteases are in the center of many diseases, and consequently, proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N termini. Methods such as combined fractional diagonal chromatography (COFRADIC)1 and, later, terminal amine isotopic labeling of substrates (TAILS) have revealed numerous insights into protease substrates and consensus motifs. We present an alternative and simple protocol for N-terminal peptide enrichment, based on charge-based fractional diagonal chromatography (ChaFRADIC) and requiring only well-established protein chemistry and a pipette tip. Using iTRAQ-8-plex, we quantified on average 2,073 ± 52 unique N-terminal peptides from only 4.3 µg per sample/channel, allowing the identification of proteolytic targets and consensus motifs. This high sensitivity may even allow working with clinical samples such as needle biopsies in the future. We applied our method to study the dynamics of staurosporine-induced apoptosis. Our data demonstrate an orchestrated regulation of specific pathways after 1.5 h, 3 h, and 6 h of treatment, with many important players of homeostasis targeted already after 1.5 h. We additionally observed an early multilevel modulation of the splicing machinery both by proteolysis and phosphorylation. This may reflect the known role of alternative splicing variants for a variety of apoptotic genes, which seems to be a driving force of staurosporine-induced apoptosis.

Protease Specificity Profiling in a Pipet Tip Using "Charge-Synchronized" Proteome-Derived Peptide Libraries.

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method's versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity.

Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases.

Rhomboid proteases form one of the most widespread intramembrane protease families. They have been implicated in variety of human diseases. The currently reported rhomboid inhibitors display some selectivity, but their construction involves multistep synthesis protocols. Here, we report benzoxazin-4-ones as novel inhibitors of rhomboid proteases with a covalent, but slow reversible inhibition mechanism. Benzoxazin-4-ones can be synthesized from anthranilic acid derivatives in a one-step synthesis, making them easily accessible. We demonstrate that an alkoxy substituent at the 2-position is crucial for potency and results in low micromolar inhibitors of rhomboid proteases. Hence, we expect that these compounds will allow rapid synthesis and optimization of inhibitors of rhomboids from different organisms.

Discovery and Biological Evaluation of Potent and Selective N-Methylene Saccharin-Derived Inhibitors for Rhomboid Intramembrane Proteases.

Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, rhomboids have been associated with multiple diseases, including Parkinson's disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against rhomboids both in vitro and in live Escherichia coli cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.

Synthesis and Application of Activity-Based Probes for Proteases.

The detection, visualization, and identification of active proteases can be facilitated by activity-based probes, which covalently bind to a catalytic residue of the target protease. The synthesis of activity-based probes can be challenging. We here outline a simple protocol for probe synthesis based on standard solid phase peptide synthesis followed by capping of the N-terminus with a reactive electrophile as a warhead. The applicability of the probes is illustrated by labeling cysteine proteases in cell and tissue lysates with Western blotting or fluorescence scanning as a readout.

Isoform-Specific Phosphorylation in Human Hsp90ß Affects Interaction with Clients and the Cochaperone Cdc37.

The 90-kDa heat shock proteins (Hsp90s) assist the maturation of many key regulators of signal transduction pathways and cellular control circuits like protein kinases and transcription factors and chaperone their stability and activity. In this function, Hsp90s cooperate with some 30 cochaperones and they are themselves subject to regulation by numerous post-translational modifications. In vertebrates, two major isoforms exist in the cytosol, Hsp90α and Hsp90ß, which share a high degree of sequence identity and are expressed in tissue- and environmental condition-dependent manner. We identified an isoform-specific phosphorylation site in human Hsp90ß. This phosphorylation site seems to be linked to vertebrate evolution since it is not found in invertebrata but in all tetrapoda and many but not all fish species. We provide data suggesting that this phosphorylation is important for the activation of Hsp90 clients like glucocorticoid receptor and a protein kinase. Replacement of the phosphorylation site by glutamate affects the conformational dynamics of Hsp90 and interaction with the kinase-specific cochaperone Cdc37.

General and Modular Strategy for Designing Potent, Selective, and Pharmacologically Compliant Inhibitors of Rhomboid Proteases.

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

Chemical Tools for the Study of Intramembrane Proteases.

Intramembrane proteases (IMPs) reside inside lipid bilayers and perform peptide hydrolysis in transmembrane or juxtamembrane regions of their substrates. Many IMPs are involved in crucial regulatory pathways and human diseases, including Alzheimer's disease, Parkinson's disease, and diabetes. In the past, chemical tools have been instrumental in the study of soluble proteases, enabling biochemical and biomedical research in complex environments such as tissue lysates or living cells. However, IMPs place special challenges on probe design and applications, and progress has been much slower than for soluble proteases. In this review, we will give an overview of the available chemical tools for IMPs, including activity-based probes, affinity-based probes, and synthetic substrates. We will discuss how these have been used to increase our structural and functional understanding of this fascinating group of enzymes, and how they might be applied to address future questions and challenges.

Differences in conformational dynamics within the Hsp90 chaperone family reveal mechanistic insights.

The molecular chaperones of the Hsp90 family are essential in all eukaryotic cells. They assist late folding steps and maturation of many different proteins, called clients, that are not related in sequence or structure. Hsp90 interaction with its clients appears to be coupled to a series of conformational changes. Using hydrogen exchange mass spectrometry (HX-MS) we investigated the structural dynamics of human Hsp90ß (hHsp90) and yeast Hsp82 (yHsp82). We found that eukaryotic Hsp90s are much more flexible than the previously studied Escherichia coli homolog (EcHtpG) and that nucleotides induce much smaller changes. More stable conformations in yHsp82 are obtained in presence of co-chaperones. The tetratricopeptide repeat (TPR) domain protein Cpr6 causes a different amide proton protection pattern in yHsp82 than the previously studied TPR-domain protein Sti1. In the simultaneous presence of Sti1 and Cpr6, protection levels are observed that are intermediate between the Sti1 and the Cpr6 induced changes. Surprisingly, no bimodal distributions of the isotope peaks are detected, suggesting that both co-chaperones affect both protomers of the Hsp90 dimer in a similar way. The cochaperones Sba1 was found previously in the crystal structure bound to the ATP hydrolysis-competent conformation of Hsp90, which did not allow to distinguish the mode of Sba1-mediated inhibition of Hsp90's ATPase activity by stabilizing the pre- or post-hydrolysis step. Our HX-MS experiments now show that Sba1 binding leads to a protection of the ATP binding lid, suggesting that it inhibits Hsp90's ATPase activity by slowing down product release. This hypothesis was verified by a single-turnover ATPase assay. Together, our data suggest that there are much smaller energy barriers between conformational states in eukaryotic Hsp90s than in EcHtpG and that co-chaperones are necessary in addition to nucleotides to stabilize defined conformational states.

The social contexts of depression during motherhood: a study of Explanatory Models in Vietnam.

BACKGROUND: Major depression is increasing world-wide, and is the fourth leading cause of the global disease burden. Depression is rarely diagnosed in primary care settings in Vietnam, and those afflicted usually only seek professional care when the illness has become very severe. Depressive disorders affecting mothers are an important cause of low birth-weight, childhood stunting, under nutrition and adverse mental development, and a study has shown a 33% prevalence of postnatal depression symptoms in Ho Chi Minh City. METHODS: The aim of this study was to elicit Illness Explanatory Models (EMs) of depression and postnatal depression from nine mothers and nine health workers. The study was conducted in a semi-rural area in Vietnam, and the EMs were elicited through semi-structured interviews where a case vignette of depression was used as the basis of questioning. RESULTS: The EMs elicited were predominantly somatosocial in nature and the mothers assigned a strong personal responsibility for care. Psychiatric treatment and care was seldom recommended. Lack of communication was described as an important factor concealing depression, and together with the lack of care-seeking can be expected to impede effective treatment. LIMITATIONS: The results of this study cannot be generalised beyond the group studied, or the context of Ba Vi, though we believe that analytical generalisation to other contexts can be made. CONCLUSION (CLINICAL RELEVANCE): The results of this study highlight the importance of depression and postnatal depression being diagnosed in primary care, and of a cross-sectoral approach for the prevention of depression in Vietnam, which takes into account the social causation of depression in women.