Structure of puromycin-sensitive aminopeptidase and polyglutamine binding.

Puromycin-sensitive aminopeptidase (E.C. 3.

Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3.

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3's oncogenic functions are not well understood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards highly homologous family members PRL-1 and PRL-2.

Aerosol capture and coronavirus spike protein deactivation by enzyme functionalized antiviral membranes.

The airborne nature of coronavirus transmission makes it critical to develop new barrier technologies that can simultaneously reduce aerosol and viral spread. Here, we report nanostructured membranes with tunable thickness and porosity for filtering coronavirus-sized aerosols, combined with antiviral enzyme functionalization that can denature spike glycoproteins of the SARS-CoV-2 virus in low-hydration environments. Thin, asymmetric membranes with subtilisin enzyme and methacrylic functionalization show more than 98.

Toxolysin 4 Contributes to Efficient Parasite Egress from Host Cells.

Egress from host cells is an essential step in the lytic cycle of T. gondii and other apicomplexan parasites; however, only a few parasite secretory proteins are known to affect this process. The putative metalloproteinase toxolysin 4 (TLN4) was previously shown to be an extensively processed microneme protein, but further characterization was impeded by the inability to genetically ablate .

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies.

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity.

Insulin-degrading enzyme is not secreted from cultured cells.

Insulin-degrading enzyme (IDE) functions in the catabolism of bioactive peptides. Established roles include degrading insulin and the amyloid beta peptide (Aβ), linking it to diabetes and Alzheimer's disease. IDE is primarily located in the cytosol, and a longstanding question is how it gains access to its peptide substrates.

Sustained Activation of Rho GTPases Promotes a Synthetic Pulmonary Artery Smooth Muscle Cell Phenotype in Neprilysin Null Mice.

Objective: Pulmonary artery smooth muscle cells (PASMCs) from neprilysin (NEP) null mice exhibit a synthetic phenotype and increased activation of Rho GTPases compared with their wild-type counterparts. Although Rho GTPases are known to promote a contractile SMC phenotype, we hypothesize that their sustained activity decreases SM-protein expression in these cells.

Approach And Results: PASMCs isolated from wild-type and NEP mice were used to assess levels of SM-proteins (SM-actin, SM-myosin, SM22, and calponin) by Western blotting, and were lower in NEP PASMCs compared with wild-type.

Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes.

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE.

Hyperamylinemia Increases IL-1β Synthesis in the Heart via Peroxidative Sarcolemmal Injury.

Hypersecretion of amylin is common in individuals with prediabetes, causes amylin deposition and proteotoxicity in pancreatic islets, and contributes to the development of type 2 diabetes. Recent studies also identified amylin deposits in failing hearts from patients with obesity or type 2 diabetes and demonstrated that hyperamylinemia accelerates the development of heart dysfunction in rats expressing human amylin in pancreatic β-cells (HIP rats). To further determine the impact of hyperamylinemia on cardiac myocytes, we investigated human myocardium, compared diabetic HIP rats with diabetic rats expressing endogenous (nonamyloidogenic) rat amylin, studied normal mice injected with aggregated human amylin, and developed in vitro cell models.

Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer's Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats.

Amylin is a hormone synthesized and co-secreted with insulin by pancreatic β-cells that crosses the blood-brain barrier and regulates satiety. Amylin from humans (but not rodents) has an increased propensity to aggregate into pancreatic islet amyloid deposits that contribute to β-cell mass depletion and development of type-2 diabetes by inducing oxidative stress and inflammation. Recent studies demonstrated that aggregated amylin also accumulates in brains of Alzheimer's disease (AD) patients, preponderantly those with type-2 diabetes.

The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro.

An Extended Polyanion Activation Surface in Insulin Degrading Enzyme.

Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber.

Reduction of amyloid-beta levels in mouse eye tissues by intra-vitreally delivered neprilysin.

Amyloid-beta (Aβ) is a group of aggregation-prone, 38- to 43-amino acid peptides generated in the eye and other organs. Numerous studies suggest that the excessive build-up of low-molecular-weight soluble oligomers of Aβ plays a role in the progression of Alzheimer's disease and other brain degenerative diseases. Recent studies raise the hypothesis that excessive Aβ levels may contribute also to certain retinal degenerative diseases.

Mercury Reduces the Enzymatic Activity of Neprilysin in Differentiated SH-SY5Y Cells.

Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer's disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1-20 μM of Hg.

Obesity and diabetes cause cognitive dysfunction in the absence of accelerated β-amyloid deposition in a novel murine model of mixed or vascular dementia.

Mid-life obesity and type 2 diabetes mellitus (T2DM) confer a modest, increased risk for Alzheimer's disease (AD), though the underlying mechanisms are unknown. We have created a novel mouse model that recapitulates features of T2DM and AD by crossing morbidly obese and diabetic db/db mice with APPΔNL/ΔNLx PS1P264L/P264L knock-in mice. These mice (db/AD) retain many features of the parental lines (e.

Neprilysin regulates pulmonary artery smooth muscle cell phenotype through a platelet-derived growth factor receptor-dependent mechanism.

Reduced neprilysin (NEP), a cell surface metallopeptidase, which cleaves and inactivates proinflammatory and vasoactive peptides, predisposes the lung vasculature to exaggerated remodeling in response to hypoxia. We hypothesize that loss of NEP in pulmonary artery smooth muscle cells results in increased migration and proliferation. Pulmonary artery smooth muscle cells isolated from NEP(-/-) mice exhibited enhanced migration and proliferation in response to serum and platelet-derived growth factor, which was attenuated by NEP replacement.

Cysteine 904 is required for maximal insulin degrading enzyme activity and polyanion activation.

Cysteine residues in insulin degrading enzyme have been reported as non-critical for its activity. We found that converting the twelve cysteine residues in rat insulin degrading enzyme (IDE) to serines resulted in a cysteine-free form of the enzyme with reduced activity and decreased activation by polyanions. Mutation of each cysteine residue individually revealed cysteine 904 as the key residue required for maximal activity and polyanion activation, although other cysteines affect polyanion binding to a lesser extent.

Active site mutations change the cleavage specificity of neprilysin.

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563) and Ser(546). Among the mutants studied in detail we observed changes in their activity towards leucine(5)-enkephalin, insulin B chain, and amyloid β(1-40).

BACE2 expression increases in human neurodegenerative disease.

β-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-β (Aβ) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: β-site Aβ precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2.

Anion activation site of insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) (insulysin) is a zinc metallopeptidase that metabolizes several bioactive peptides, including insulin and the amyloid β peptide. IDE is an unusual metallopeptidase in that it is allosterically activated by both small peptides and anions, such as ATP. Here, we report that the ATP-binding site is located on a portion of the substrate binding chamber wall arising largely from domain 4 of the four-domain IDE.

Identification of the allosteric regulatory site of insulysin.

Background: Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP.

Principal Findings: The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2.

Mixed dimers of insulin-degrading enzyme reveal a cis activation mechanism.

Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity.

Catalase-coupled gold nanoparticles: comparison between the carbodiimide and biotin-streptavidin methods.

The use of proteins for therapeutic applications requires the protein to maintain sufficient activity for the period of in vivo treatment. Many proteins exhibit a short half-life in vivo and, thus, require delivery systems for them to be applied as therapeutics. The relative biocompatibility and the ability to form functionalized bioconjugates via simple chemistry make gold nanoparticles excellent candidates as protein delivery systems.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity.

Aminopeptidases do not directly degrade tau protein.

Background: Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA).