Impact of Protein N-Modifications on Cellular Functions and Human Health.

Most human proteins are modified by enzymes that act on the α-amino group of a newly synthesized polypeptide. Methionine aminopeptidases can remove the initiator methionine and expose the second amino acid for further modification by enzymes responsible for myristoylation, acetylation, methylation, or other chemical reactions. Specific acetyltransferases can also modify the initiator methionine and sometimes the acetylated methionine can be removed, followed by further modifications.

Proteomic Analysis of Human Immune Responses to Live-Attenuated Tularemia Vaccine.

() is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%.

Comparison of a Novel Homogeneous Cyclic Amp Assay and a Luciferase Assay for Measuring Stimulating Thyrotropin-Receptor Autoantibodies.

Objective: Stimulating thyrotropin-receptor antibodies (TSAb) cause Graves' disease (GD). We tested a novel homogeneous fluorescent 3',5' cyclic adenine monophosphate (cAMP) assay for the detection of TSAb in a bioassay.

Methods: Chinese hamster ovary (CHO) cell lines expressing either a chimeric (MC4) or wild-type (WT) TSH-R were incubated with the adenyl cyclase activator forskolin, a human TSAb monoclonal antibody (M22), and with sera from GD patients.

Common therapeutic target for both cancer and obesity.

Obesity and cancer are two interrelated conditions of high epidemiological need, with studies showing that obesity is responsible for nearly 25% of the relative contribution to cancer incidence. Given the connection between these conditions, a drug that can operate on both obesity and cancer is highly desirable. Such a drug is accomplishable through the development of potent anti-angiogenesis agents due to the shared underlying role of angiogenesis in the development of both diseases.

A pilot study of pepsin in tracheal and oral secretions.

Background: Because reflux of gastric juice into the oropharynx must precede its aspiration into the lungs, it is reasonable to hypothesize that the detection of pepsin (the major gastric enzyme in gastric juice) in oral secretions may provide a relatively noninvasive method of predicting risk for aspiration.

Objective: To describe the incidence of pepsin in oral and tracheal secretions collected concurrently from a sample of 50 gastric-fed patients undergoing mechanical ventilation.

Methods: An exploratory descriptive design with a convenience sample from 4 medical and surgical intensive care units.

Molecular PINCERs® for biomarker analysis and their potential application in hepatitis C diagnosis.

Molecular PINCERs(®) are antibody-based protein sensors that allow rapid and sensitive detection of specific proteins in solution. The sensors allow quick detection of a target, with sensitivity and specificity approaching that of conventional immunoassays, but require no sample manipulation other than mixing of the test sample with sensors. Combining the PINCER assay with immunoprecipitation allows quick and sensitive detection of anti-HCV immunoglobulin (Ig)G and IgM in human serum.

A homogeneous fluorescent sensor for human serum albumin.

Human serum albumin is the most abundant protein in the body and is an important biomarker used for disease-related diagnosis. Although the traditional enzyme-linked immunosorbent assay (ELISA) approach can precisely measure the concentration of human serum albumin, the multi-step procedure and time-consuming preparations of ELISA limit its diagnostic applications, preventing accurate point-of-care testing, for example. Herein, we report the recent development of an antibody-based albumin sensor that allows for a homogeneous measurement of albumin concentrations in saliva, urine and serum, in which this type of sensor is validated for the first time.

A homogeneous fluorescent assay for cAMP-phosphodiesterase enzyme activity.

Cyclic adenosine monophosphate-phosphodiesterases (cAMP-PDEs) regulate the cellular level of cAMP by selectively catalyzing the hydrolysis of the phosphodiester bond in the cAMP molecule. They play important roles in modulating cellular and physiological functions. There is a growing interest in the study of cAMP-PDEs as therapeutic targets.

Molecular pincers: antibody-based homogeneous protein sensors.

We describe here a new homogeneous antibody-based protein sensor design (molecular pincers) that allows rapid and sensitive detection of a specific protein in solution. In the presence of the target protein these sensors produce fluorescence signal derived from target-dependent annealing of short complementary fluorochrome-labeled oligonucleotides attached to a pair of target-specific antibodies via nanometer-scale flexible linkers. The sensors allow near-instantaneous detection of the target with sensitivity and specificity approaching that of enzyme-linked immunosorbent assay (ELISA) but requiring no sample manipulation other then the addition of the sample to the sensor mix.

Profiling constitutive proteolytic events in vivo.

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis.

Molecular beacons for DNA binding proteins: an emerging technology for detection of DNA binding proteins and their ligands.

Quantitation of the level or activity of specific proteins is one of the most commonly performed experiments in biomedical research. Protein detection has historically been difficult to adapt to high throughput platforms because of heavy reliance upon antibodies for protein detection. Molecular beacons for DNA binding proteins is a recently developed technology that attempts to overcome such limitations.

Stellate cell apoptosis by a soluble mediator from immortalized human hepatocytes.

Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix in fibrosis and cirrhosis. In this study, we have investigated the role of hepatitis C virus (HCV) core protein induced immortalized human hepatocytes (IHH) on HSC growth. Preferential growth of IHH and apoptosis of activated human hepatic stellate cells (LX2) were observed upon coculture of these two cell types in a dual chamber or in the presence of conditioned medium (CM) from IHH.

Tracheobronchial aspiration of gastric contents in critically ill tube-fed patients: frequency, outcomes, and risk factors.

Objectives: To describe the frequency of pepsin-positive tracheal secretions (a proxy for the aspiration of gastric contents), outcomes associated with aspiration (including a positive Clinical Pulmonary Infection Score [a proxy for pneumonia] and use of hospital resources), and risk factors associated with aspiration and pneumonia in a population of critically ill tube-fed patients.

Design: Prospective descriptive study conducted over a 2-yr period.

Setting: Five intensive care units in a university-affiliated medical center with level I trauma status.

ATP hydrolysis-dependent disassembly of the 26S proteasome is part of the catalytic cycle.

ATP hydrolysis is required for degradation of polyubiquitinated proteins by the 26S proteasome but is thought to play no role in proteasomal stability during the catalytic cycle. In contrast to this view, we report that ATP hydrolysis triggers rapid dissociation of the 19S regulatory particles from immunopurified 26S complexes in a manner coincident with release of the bulk of proteasome-interacting proteins. Strikingly, this mechanism leads to quantitative disassembly of the 19S into subcomplexes and free Rpn10, the polyubiquitin binding subunit.

Yeast glutamine-fructose-6-phosphate aminotransferase (Gfa1) requires methionine aminopeptidase activity for proper function.

Methionine aminopeptidase (MetAP) catalyzes the co-translational processing of initiator methionine from nascent proteins. A cellular requirement for MetAP activity is likely due to dysfunction of MetAP substrates that require methionine removal for proper protein function. Glutamine-fructose-6-phosphate aminotransferase (Gfa1) is an essential enzyme in yeast that catalyzes the first and rate-limiting step in hexosamine biosynthesis.

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae.

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro.

Detection of pepsin in tracheal secretions after forced small-volume aspirations of gastric juice.

Background: Detecting small-volume aspirations of gastric contents is an important but difficult task. A potentially useful method for this purpose is assaying tracheal secretions for pepsin, an expected constituent of gastric juice.

Methods: A 2-group experimental design was used.

N-terminal methionine removal and methionine metabolism in Saccharomyces cerevisiae.

Methionine aminopeptidase (MetAP) catalyzes removal of the initiator methionine from nascent polypeptides. In eukaryotes, there are two forms of MetAP, type 1 and type 2, whose combined activities are essential, but whose relative intracellular roles are unclear. Methionine metabolism is an important aspect of cellular physiology, involved in oxidative stress, methylation, and cell cycle.

Functional expression of human methionine aminopeptidase type 1 in Saccharomyces cerevisiae.

We expressed recombinant human methionine aminopeptidase type 1 (MAP or MetAP) in a map1 null yeast strain to determine the extent of functional complementation between the two proteins. The human MetAP1 protein fully rescued the slow growth phenotype associated with deletion of yeast MetAP1, suggesting that the yeast and human MetAP1 proteins may have similar roles in vivo. Expression of human MetAP1 in yeast has significance in understanding the function of the human protein, studying its in vivo substrate specificity, and developing specific anti-fungal drugs to target yeast MetAP1.

Yeast methionine aminopeptidase type 1 is ribosome-associated and requires its N-terminal zinc finger domain for normal function in vivo.

Methionine aminopeptidase type 1 (MetAP1) cotranslationally removes N-terminal methionine from nascent polypeptides, when the second residue in the primary structure is small and uncharged. Eukaryotic MetAP1 has an N-terminal zinc finger domain not found in prokaryotic MetAPs. We hypothesized that the zinc finger domain mediates the association of MetAP1 with the ribosomes and have reported genetic evidence that it is important for the normal function of MetAP1 in vivo.

Pepsin as a marker for pulmonary aspiration.

Background: Although assessment for aspiration of small volumes of gastric contents in tube-fed patients receiving mechanical ventilation is important, available methods for this purpose are not wholly satisfactory. A potential method is immunoassay of tracheal secretions for the gastric enzyme pepsin.

Objectives: To determine the frequency with which pepsin in suctioned tracheal secretions from acutely ill, tube-fed patients receiving mechanical ventilation could be detected via an immunoassay.

The specificity in vivo of two distinct methionine aminopeptidases in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential function of amino-terminal methionine removal is provided cotranslationally by two methionine aminopeptidases (MetAP1 and MetAP2). To examine the individual processing efficiency of each MetAP in vivo, we measured the degree of N-terminal methionine cleavage from a series of mutated glutathione-S-transferase (GST) proteins isolated from yeast wild-type, a map1 deletion strain, a map2 deletion strain, and a map1 deletion strain overexpressing the MAP2 gene. We found that MetAP1 plays the major role in N-terminal methionine removal in yeast.

5-demethylovalicin, as a methionine aminopeptidase-2 inhibitor produced by Chrysosporium.

5-Demethylovalicin was isolated from the fermentation broth Chrysosporium lucknowense and the structure was identified by spectroscopic methods. 5-Demethylovalicin inhibited the recombinant human MetAP-2 (IC(50)=17.7 nM) and the growth of human umbilical vein endothelial cells (HUVEC; IC(50)=100 nM) in cell proliferation assay without cytotoxicity on the transformed and cancer cell lines.