Prolyl endopeptidase activity in bronchoalveolar lavage fluid: a novel diagnostic biomarker in a guinea pig model of invasive pulmonary aspergillosis.

Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.

3D Spheroids of Human Primary Urine-Derived Stem Cells in the Assessment of Drug-Induced Mitochondrial Toxicity.

Mitochondrial toxicity (Mito-Tox) risk has increased due to the administration of several classes of drugs, particularly some life-long antiretroviral drugs for HIV+ individuals. However, no suitable in vitro assays are available to test long-term Mito-Tox (≥4 weeks). The goal of this study is to develop a 3D spheroid system of human primary urine-derived stem cells (USC) for the prediction of drug-induced delayed Mito-Tox. The cytotoxicity and Mito-Tox were assessed in 3D USC spheroids 4 weeks after treatment with antiretroviral drugs: zalcitabine (ddC; 0.1, 1 and 10 µM), tenofovir (TFV; 3, 30 and 300 µM) or Raltegravir (RAL; 2, 20 and 200 µM). Rotenone (RTNN, 10 µM) and 0.1% DMSO served as positive and negative controls. Despite only mild cytotoxicity, ddC significantly inhibited the expression of oxidative phosphorylation enzyme Complexes I, III, and IV; and RAL transiently reduced the level of Complex IV. A significant increase in caspase 3 and ROS/RNS level but a decrease in total ATP were observed in USC treated with ddC, TFV, RAL, and RTNN. Levels of mtDNA content and mitochondrial mass were decreased in ddC but minimally or not in TFV- and RAL-treated spheroids. Thus, 3D USC spheroid using antiretroviral drugs as a model offers an alternative platform to assess drug-induced late Mito-Tox.

Mixing and matching detergents for membrane protein NMR structure determination.

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

Site-specific Substitutions Eliminate Aggregation Properties of Hemopressin.

Hemopressin is a naturally occurring and therapeutically relevant peptide with applications in hypertension, pain, addiction, and obesity. We had previously demonstrated that hemopressin converts into amyloid-like fibrils under aqueous conditions. However, the amino acid residues that modulate the aggregation propensity of hemopressin were not identified. In this study, we designed and synthesized 25 different analogs of hemopressin and analyzed their aggregation properties using the principle of dynamic light scattering. As a result, we were able to identify four conservative changes in the peptide sequence (Val(2) →DVal(2), Asn(3) →Gln(3) Leu(7) →Npg(7) and C-OH→C-NH2) that minimize aggregation propensity of hemopressin. The results indicate that hemopressin aggregation is cooperative in nature and involves contribution from multiple amino acids within the peptide chain. The analogs and the corresponding aggregation propensity data reported in this study would be useful for researchers investigating therapeutic properties of hemopressin, which have been hampered due to the tendency of hemopressin to aggregate in aqueous solutions.

Preclinical Evaluations To Identify Optimal Linezolid Regimens for Tuberculosis Therapy.

UNLABELLED: Linezolid is an oxazolidinone with potent activity against Mycobacterium tuberculosis. Linezolid toxicity in patients correlates with the dose and duration of therapy. These toxicities are attributable to the inhibition of mitochondrial protein synthesis. Clinically relevant linezolid regimens were simulated in the in vitro hollow-fiber infection model (HFIM) system to identify the linezolid therapies that minimize toxicity, maximize antibacterial activity, and prevent drug resistance. Linezolid inhibited mitochondrial proteins in an exposure-dependent manner, with toxicity being driven by trough concentrations. Once-daily linezolid killed M. tuberculosis in an exposure-dependent manner. Further, 300 mg linezolid given every 12 hours generated more bacterial kill but more toxicity than 600 mg linezolid given once daily. None of the regimens prevented linezolid resistance. These findings show that with linezolid monotherapy, a clear tradeoff exists between antibacterial activity and toxicity. By identifying the pharmacokinetic parameters linked with toxicity and antibacterial activity, these data can provide guidance for clinical trials evaluating linezolid in multidrug antituberculosis regimens. IMPORTANCE: The emergence and spread of multidrug-resistant M. tuberculosis are a major threat to global public health. Linezolid is an oxazolidinone that is licensed for human use and has demonstrated potent activity against multidrug-resistant M. tuberculosis. However, long-term use of linezolid has shown to be toxic in patients, often resulting in thrombocytopenia. We examined therapeutic linezolid regimens in an in vitro model to characterize the exposure-toxicity relationship. The antibacterial activity against M. tuberculosis was also assessed for these regimens, including the amplification or suppression of resistant mutant subpopulations by the chosen regimen. Higher exposures of linezolid resulted in greater antibacterial activity, but with more toxicity and, for some regimens, increased resistant mutant subpopulation amplification, illustrating the trade-off between activity and toxicity. These findings can provide valuable insight for designing optimal dosage regimens for linezolid that are part of the long combination courses used to treat multidrug-resistant M. tuberculosis.

Sample collection in clinical proteomics--proteolytic activity profile of serum and plasma.

PURPOSE: Proteolytic enzymes are promising diagnostic targets since they play key roles in diverse physiological processes and have been implicated in numerous human diseases. Human blood is a relatively noninvasive source for disease-specific protease biomarker detection and subsequent translation into diagnostic tests. However, the choice of serum or plasma, and more specifically, which anticoagulant to choose in plasma preparation, is important to address in the sample preparation phase of biomarker discovery. EXPERIMENTAL DESIGN: We have previously utilized a combinatorial library of internally quenched fluorogenic probes to successfully map the global proteolytic profiles of various biological fluids. In this study, we utilized the platform to ascertain the impact of three commonly used anticoagulants (EDTA, heparin, and citrate) on the proteolytic activity profile of plasma and serum collected from a healthy Caucasian male. RESULTS: Serum and plasma citrate were observed to be most proteolytically active, followed by plasma heparin and then plasma EDTA. Detailed analysis of the amino acid distribution of motifs cleaved and not cleaved by the samples offered significant insights in to active proteolytic components within them. CONCLUSION AND CLINICAL RELEVANCE: Broad quantitative comparison of proteolytic profiles of these samples revealed several novel insights related to the differential substrate recognition of proteases present in these biological fluids.

Design of a serum stability tag for bioactive peptides.

Serum has a high intrinsic proteolytic activity that leads to continuous processing of peptides and proteins. Strategies to protect bioactive peptides from serum proteolytic degradation include incorporation of unnatural amino acids, conformational constraints, large polymeric tags, or other synthetic manipulations such as amide bond replacements. Here we explored a possibility of designing a serum stability tag made of natural amino acids. We observed that a diproline motif (-Pro-Pro-) shows remarkable stability against serum endopeptidases. Accordingly, we designed close to 50 peptides to identify natural amino acids flanking the -Pro-Pro- sequence that can enhance the serum stability of this motif. As a result, a tetrapeptide with the sequence Asp-Pro-Pro-Glu (DPPE) was identified that remains intact in human serum for more than 24 h. at 37°C.

Proteolytic fingerprinting of complex biological samples using combinatorial libraries of fluorogenic probes.

Proteases have garnered interest as candidate biomarkers and therapeutic targets for many human diseases. A key challenge is the identification and characterization of disease-relevant proteases in the complex milieu of biological fluids such as serum, plasma, and bronchoalveolar lavage, in which a multitude of hydrolases act in concert. This unit describes a protocol to map the global proteolytic substrate specificities of complex biological samples using a concise combinatorial library of internally quenched fluorogenic peptide probes (IQFPs). This substrate profiling approach provides a global and quantitative comparison of protease specificities between different biological samples. Such a comparative analysis can lead to the identification of disease-specific 'fingerprints' of proteolytic activities with potential utility in diagnosis and therapy.

Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α.

Post-proline cleaving peptidases are promising therapeutic targets for neurodegenerative diseases, psychiatric conditions, metabolic disorders, and many cancers. Prolyl oligopeptidase (POP; E.C. 3.4.21.26) and fibroblast activation protein α (FAP; E.C. 3.4.24.B28) are two post-proline cleaving endopeptidases with very similar substrate specificities. Both enzymes are implicated in numerous human diseases, but their study is impeded by the lack of specific substrate probes. We interrogated a combinatorial library of proteolytic substrates and identified novel and selective substrates of POP and FAP. These new sequences will be useful as probes for fundamental biochemical study, scaffolds for inhibitor design, and triggers for controlled drug delivery.

Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases.

Proteases are candidate biomarkers and therapeutic targets for many diseases. Sensitive and robust techniques are needed to quantify proteolytic activities within the complex biological milieu. We hypothesized that a combinatorial protease substrate library could be used effectively to identify similarities and differences between serum and bronchoalveolar lavage fluid (BALF), two body fluids that are clinically important for developing targeted therapies and diagnostics. We used a concise library of fluorogenic probes to map the protease substrate specificities of serum and BALF from guinea pigs. Differences in the proteolytic fingerprints of the two fluids were striking: serum proteases cleaved substrates containing cationic residues and proline, whereas BALF proteases cleaved substrates containing aliphatic and aromatic residues. Notably, cleavage of proline-containing substrates dominated all other protease activities in both human and guinea pig serum. This substrate profiling approach provides a foundation for quantitative comparisons of protease specificities between complex biological samples.

Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus.

Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.

Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.

BACKGROUND: The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers. METHODOLOGY AND PRINCIPAL FINDINGS: As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures. CONCLUSIONS: This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.