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.

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.

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods.

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.

Design of a peptide inhibitor of tyrosine kinase 2.

Tyrosine kinase inhibitors show great promise as clinical therapies, but small molecule inhibitors that are available in the clinic and under development bind to the adenosine triphosphate binding domain of the kinase, potentially limiting efficacy and selectivity. The development of antisense peptide inhibitors is a relatively unexplored area of research, and here we investigate inhibitory peptides specific for the Janus-associated kinase (JAK) family member, tyrosine kinase 2 (TYK2). We have developed peptides that are 2-3 times more selective for TYK2 than other JAK family members, with a TYK2 IC50 of 1.

In silico systems biology approaches for the identification of antimicrobial targets.

Classical antibiotic discovery efforts have relied mainly on molecular library screening coupled with target-based lead optimization. The conventional approaches are unable to tackle the emergence of antibiotic resistance and are failing to provide understanding of multiple mechanisms behind drug actions and the off-target effects. These insufficiencies have prompted researchers to focus on a multidisciplinary approach of systems biology-based antibiotic discovery.

Serum biomarkers reveal long-term cardiac injury in isoproterenol-treated African green monkeys.

The assessment of cardiac toxicity is a major challenge in both drug development and clinical trials, and numerous marketed pharmaceuticals have been removed from the market due to unpredicted cardiac effects. Serum troponins are widely used indicators of cardiac injury; however, they are short-lived and have not been validated in preclinical animal models. In this study, we have used filter-aided sample preparation (FASP) and tandem mass tag (TMT) labeling to investigate serum protein alterations in isoproterenol-treated African green monkeys.

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.

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).

Modulation of the cannabinoid receptors by hemopressin peptides.

Changes in the endocannabinoid system are implicated in numerous diseases, making it an attractive target for pharmaceutical development. The endocannabinoid receptors have traditionally been thought to act through the effects of lipophilic messengers called cannabinoids. The exciting finding of endocannabinoid system modulation by the nonapeptide hemopressin and its N-terminal extensions has highlighted the complexity of cannabinoid biology and pharmacology and sparked interest for therapeutic purposes.

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.

The ST Pinch: A side chain-to-side chain hydrogen-bonded motif.

The ST Pinch is a 12-membered hydrogen-bonded motif (Ser/Thr-Xaa-Ser/Thr) involving the side chain oxygen atoms of two Ser/Thr residues. We identified the ST Pinch in 104 proteins in a database containing high-resolution crystal structures. Conformational analysis of the ST Pinch in these proteins points to specific preferences for the Xaa residue and a high propensity of this residue to adopt positive φ angles.

Hemopressin forms self-assembled fibrillar nanostructures under physiologically relevant conditions.

The nonapeptide hemopressin, which is derived from the α chain of hemoglobin, has been reported to exhibit inverse agonist activity against the CB1 receptor. Administration of this peptide in animal models led to decreased food intake and elicited hypotensive and antinociceptive effects. On the basis of hemopressin's potential in therapeutic applications and the lack of a structure-activity relationship study in literature, we aimed to determine the conformational features of hemopressin under physiological conditions.

The serine-proline turn: a novel hydrogen-bonded template for designing peptidomimetics.

Serine-Proline (SP) dipeptide motifs have been shown to form unique hydrogen-bonding patterns in protein crystal structures. Peptides were designed to mimic these patterns by forming the 6 + 10 and the 9 + 10 hydrogen-bonded rings. Factors that contribute to the formation of SP turns include controlling backbone flexibility and amino acid chirality along with creating a hydrophobic environment around the intramolecular hydrogen bonds.

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.

An enhanced β turn in water.

Aiming to design short linear peptides featuring strong intramolecular hydrogen bonds in water, a series of tetrapeptides based on the sequence Ac-Ala-Pro-Ala-Ala-NH(2) containing all possible combinations of L- and D-amino acids was synthesized. A regiospecific combination of heterochiral residues (DDLL or its mirror image LLDD) can be used to increase turn formation and stability within short peptides in water.

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.

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 virulence of the opportunistic fungal pathogen Aspergillus fumigatus requires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR).

The filamentous fungal pathogen Aspergillus fumigatus secretes hydrolytic enzymes to acquire nutrients from host tissues. The production of these enzymes exerts stress on the endoplasmic reticulum (ER), which is alleviated by two stress responses: the unfolded protein response (UPR), which adjusts the protein folding capacity of the ER, and ER-associated degradation (ERAD), which disposes of proteins that fail to fold correctly. In this study, we examined the contribution of these integrated pathways to the growth and virulence of A.

Repurposing FDA-approved drugs to combat drug-resistant Acinetobacter baumannii.

Objective: The rising occurrence of drug-resistant pathogens accentuates the need to identify novel antibiotics. We wanted to identify new scaffolds for drug discovery by repurposing FDA-approved drugs against Acinetobacter baumannii, an emerging Gram-negative nosocomial drug-resistant pathogen.

Materials And Methods: In this study, we screened 1040 FDA-approved drugs against drug-susceptible A.

Design of short linear peptides that show hydrogen bonding constraints in water.

Using a combination of an aromatic amino acid, a homoserine side chain, and a d-amino acid, a series of linear tetrapeptides were designed that adopt an "Hse turn" in water. The conformation was stabilized by intramolecular hydrogen bonds even in the presence of surrounding water molecules. In particular, the peptide with sequence H-Abz-Homoser-Ser-d-Gln-NH(2) showed significant through-space interactions and its free energy of folding is estimated to be on the order of -4 kcal/mol.

Enzyme-targeted fluorescent imaging probes on a multiple antigenic peptide core.

Peptide dendrimers have a variety of applications in biology such as the vehicles for drug and gene delivery, molecular inhibitors, protein mimics, and synthetic vaccines. The multiple antigenic peptide (MAP) system is a well-known example of a discrete, dendrimeric scaffold. We explored a novel application of the MAP-based scaffold by designing molecular probes that fluoresce only after enzymatic treatment.

Fluorescence probe with a pH-sensitive trigger.

Acid-catalyzed hydrolysis was used as the mechanism to design a new type of environmentally sensitive fluorescence probe. A mild and selective periodate oxidation of the 2-amino alcohol of serine in the presence of a disulfide bond was developed to prepare dialdehyde peptides. Two identical fluorochrome hydrazide derivatives were then linked to the dialdehyde peptide forming an acid-labile hydrazone linkage.

Potent inhibitors of LXXLL-based protein-protein interactions.

Protein-protein interactions between estrogen receptors, ERalpha and ERbeta, and their coactivators (CoAs) are an attractive target for drug intervention. This interaction is mediated by a small pentapeptide motif (LXXLL), termed the NR box. Based on this motif, a variety of cyclic and linear peptides were synthesized in order to gain a better understanding of the association of CoA proteins with the ER isoforms.