Impact of Protein Nα-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. These modifications at the protein N-termini play critical roles in cellular protein localization, protein-protein interaction, protein-DNA interaction, and protein stability. Consequently, the dysregulation of these modifications could significantly change the development and progression status of certain human diseases. The focus of this review is to highlight recent progress in our understanding of the roles of these modifications in regulating protein functions and how these enzymes have been used as potential novel therapeutic targets for various human diseases.

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

Francisella tularensis (F. tularensis) is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. F. tularensis can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%. To study the host responses to a live-attenuated tularemia vaccine, peripheral blood mononuclear cell (PBMC) samples were assayed from 10 subjects collected pre- and post-vaccination, using both the 2D-DIGE/MALDI-MS/MS and LC-MS/MS approaches. Protein expression related to antigen processing and presentation, inflammation (PPARγ nuclear receptor), phagocytosis, and gram-negative bacterial infection was enriched at Day 7 and/or Day 14. Protein candidates that could be used to predict human immune responses were identified by evaluating the correlation between proteome changes and humoral and cellular immune responses. Consistent with the proteomics data, parallel transcriptomics data showed that MHC class I and class II-related signals important for protein processing and antigen presentation were up-regulated, further confirming the proteomic results. These findings provide new biological insights that can be built upon in future clinical studies, using live attenuated strains as immunogens, including their potential use as surrogates of protection.

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. Intracellular cAMP levels were measured using a Bridge-It® cAMP assay, and the results were compared with a luciferase-based bioassay. RESULTS: Both cell lines were stimulated with forskolin concentrations (0.006-200 µM) in a dose-dependent manner. The linear range in the MC4 and WT cells was 0.8-25 and 3.1-50 µM, respectively. Levels of cAMP and luciferase in forskolin-treated MC4 and WT cells were positively correlated (r = 0.91 and 0.84, both p < 0.001). The 50% maximum stimulatory concentration of forskolin was more than 16-fold higher for the CHO-WT cells than the CHO-MC4 cells in the cAMP assay and 4-fold higher in the luciferase assay. Incubation of both cell lines with M22 (0.006-50 ng/mL) resulted in a dose-dependent increase in cAMP levels with linear ranges for the MC4 and WT cells of 0.8-12.5 and 0.2-3.125 ng/mL, respectively. Comparison of cAMP and luciferase levels in M22-treated MC4 and WT cells also showed a positive correlation (r = 0.88, p < 0.001 and 0.75, p = 0.002). A positive correlation was also noted when using patient samples (r = 0.96, p < 0.001) that were all TSH-R-Ab binding assay positive. CONCLUSION: The novel, rapid, simple-to-perform cAMP assay provides TSAb-mediated stimulatory results comparable to a luciferase-based bioassay.

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. Prior research has demonstrated a key role of type-2 methionine aminopeptidase (MetAP2) for angiogenesis, which has led to the development of numerous of novel inhibitors. Several irreversible MetAP2 inhibitors have entered clinical trials without great success. Though this lack of success could be attributed to off-target adverse effects, the underlying causes remain unclear. More promising reversible inhibitors have been recently developed with excellent pre-clinical results. However, due to insufficient knowledge of the biological functions of N-terminal protein processing, it is hard to predict whether these novel inhibitors would successfully pass clinical trials and thereby benefit cancer and obesity patients. Significantly more efforts are needed to advance our understanding of the regulation of methionine aminopeptidases and the processes by which they govern the function of proteins.

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. An oral secretion and a tracheal secretion were collected concurrently from each patient (yielding a sample of 50 oral and 50 tracheal secretions). The tracheal secretions were obtained via the inline suction system with an attached sputum trap; oral secretions were obtained via a Yankauer suction tip with an attached sputum trap. All specimens were assayed for pepsin by the Western blot method. RESULTS: Oral secretions from 10 patients (20%) and tracheal secretions from 2 patients (4%) were pepsin-positive. Both patients with pepsin-positive tracheal secretions also had pepsin-positive oral secretions. Pepsin was not found in the tracheal secretions from the remaining 8 patients with pepsin-positive oral secretions. CONCLUSIONS: Although reflux of gastric juice into the oropharynx must precede its aspiration into the lungs, individual reflux events do not necessarily lead to aspiration. Thus, it is reasonable that we found pepsin 5 times more often in oral secretions than in tracheal secretions.

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. The assay only requires simple mixing, and relies on time-resolved (TR) fluorescence resonance energy transfer (FRET) to produce robust, sensitive signals. The whole process, from sample preparation to final read-out, is expected to take less than 1h and requires only a standard plate-reader, thus making the sensor a convenient and cost-effective tool for albumin analysis.

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. This improvement is achieved through binding of anti-HCV IgG and IgM from serum to HCV-antigen-immobilized microspheres, followed by quantification of the IgG and IgM using molecular PINCERs. The PINCER-based assay is cost-effective, sensitive and has potential for clinical diagnostic use.

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. We describe a novel method for measuring the enzyme activity of cAMP-PDEs that is based on a homogeneous fluorescence assay employing a cAMP-dependent DNA-binding protein (CAP). We demonstrate that the assay is quick and robust compared to traditional methods and is expected to be cost-effective for high-throughput screening of cAMP-PDE inhibitors. The usefulness of the assay is demonstrated by measuring IC(50) values of three nonselective PDE inhibitors and by kinetic measurements of cAMP-PDEs from various rat tissues.

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. We used cardiac troponin I and C-reactive protein as the targets to validate these desirable properties of the sensors. Due to the availability of antibodies to thousands of interesting targets and the straightforward design blueprint of the sensors we expect their wide-ranging applications in research and medical diagnosis, especially when simplicity, high throughput, and short detection time are essential.

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. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.

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. Protein detection is accomplished using inexpensive, easy-to-synthesize oligonucleotides, accompanied by a fluorescence readout. Importantly, detection of the protein and reporting of the signal occur simultaneously, allowing for one-step protocols and increased potential for use in high throughput analysis. While the initial iteration of the technology allowed only for the detection of sequence-specific DNA binding proteins, more recent adaptations allow for the possibility of development of beacons for any protein, independent of native DNA binding activity. Here, we discuss the development of the technology, the mechanism of the reaction, and recent improvements and modifications made to improve the assay in terms of sensitivity, potential for multiplexing, and broad applicability.

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. CM did not display a growth inhibitory role on other hepatic (Huh-7, HepG2, Hep3B and THLE) and non-hepatic (HeLa, MCF-7, and BHK) epithelial cells, indicating that the soluble mediator from IHH does not have a generalized effect on cell lines examined in our study. Further studies suggested that CM from IHH increased the expression of TRAIL receptors on LX2 cell surface, and induced apoptosis by a caspase dependent mechanism. Peptide mass fingerprinting of the purified soluble mediator from CM suggested that gelsolin fragments may play a role in apoptosis of LX2 cells. Taken together, our results suggested that a soluble mediator secreted from immortalized human hepatocytes plays an important role in hepatic stellate cell growth regulation.

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. PATIENTS: Each of the 360 adult patients participated for 4 days. Among the inclusion criteria were mechanical ventilation and tube feedings. An exclusion criterion was physician-diagnosed pneumonia at the time of enrollment. INTERVENTION: None. MEASUREMENTS AND MAJOR RESULTS: Almost 6,000 tracheal secretions collected during routine suctioning were assayed for pepsin; of these, 31.3% were positive. At least one aspiration event was identified in 88.9% (n = 320) of the participants. The incidence of pneumonia (as determined by the Clinical Pulmonary Infection Score) increased from 24% on day 1 to 48% on day 4. Patients with pneumonia on day 4 had a significantly higher percentage of pepsin-positive tracheal secretions than did those without pneumonia (42.2% vs. 21.1%, respectively; p < .001). Length of stay in the intensive care unit and need for ventilator support were significantly greater for patients with pneumonia (p < .01). A low backrest elevation was a risk factor for aspiration (p = .024) and pneumonia (p = .018). Other risk factors for aspiration included vomiting (p = .007), gastric feedings (p = .009), a Glasgow Coma Scale score <9 (p = .021), and gastroesophageal reflux disease (p = .033). The most significant independent risk factors for pneumonia were aspiration (p < .001), use of paralytic agents (p = .002), and a high sedation level (p = .039). CONCLUSIONS: Aspiration of gastric contents is common in critically ill tube-fed patients and is a major risk factor for pneumonia. Furthermore, it leads to greater use of hospital resources. Modifiable risk factors for aspiration need to be addressed.

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. Biochemical reconstitution with purified Sic1, a prototype substrate of the Cdc34/SCF ubiquitin ligase, suggests that substrate degradation is essential for triggering the ATP hydrolysis-dependent dissociation and disassembly of the 19S and that this mechanism leads to release of degradation products. This is the first demonstration that a controlled dissociation of the 19S regulatory particles from the 26S proteasome is part of the mechanism of protein degradation.

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. The alpha-amino group of Gfa1 Cys-1 has been proposed to act as a nucleophile in the catalytic mechanism. We used two mutational strategies to evaluate whether removal of initiator methionine, catalyzed by MetAP, is required for Gfa1 function. Our results demonstrate that exposure of the alpha-amino group of Cys-1 is required for normal Gfa1 function as failure to do so results in decreased enzyme activity and slow growth. Further, either isoform of MetAP in yeast is sufficient for Gfa1 processing in vivo. These results are the first demonstration of an endogenous yeast protein that requires the exposure of the alpha-amino group by MetAP action for normal function. Additionally, Gfa1 will be a relevant target in therapeutic or physiological applications in which MetAP activity is inhibited.

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. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.

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. The primary subjects were 161 experimental and 21 control New Zealand white rabbits; 161 acutely ill humans provided the gastric juice used in the project. The animals were anesthetized before being intubated and mechanically ventilated. Three separate boluses of human gastric juice mixed with dye-stained enteral formula were instilled into the experimental animals' tracheas; the 21 control animals received only 0.9% sodium chloride solution. At the beginning of each experiment, 0.4 mL/kg of the substance was infused over a 30-minute period; the infusion was then stopped and 90 minutes were allowed to elapse before endotracheal suctioning was performed. This procedure was repeated at hour 2 and hour 4. After completion of the multiple aspiration portion of the study, 23 additional animals were subjected to a single aspiration of 0.4 mL/kg of a mixture of human gastric juice and dye-stained enteral formula; secretions were obtained at 2 hours, 4 hours, and 6 hours. An immunoassay was used to test for pepsin in all of the tracheal secretions. RESULTS: In the 3-aspiration group, pepsin was found in all of the secretions from 92.5% (149/161) of the experimental animals; in contrast, no pepsin was found in any of the secretions from the 20 control animals. In the single-aspiration group, pepsin was found in all of the tracheal secretions from the 23 animals at 2 hours and 4 hours and 21 of the 23 animals at 6 hours. CONCLUSIONS: The immunoassay used in this animal model study was able to detect pepsin in >90% of the experimental animals' tracheal secretions after multiple or single forced aspirations of gastric juice. The extent to which pepsin can be detected in the tracheal secretions of acutely ill tube-fed humans requires investigation, as does the extent to which clinical outcomes are affected by pepsin-positive tracheal secretions.

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. Due to the potential of MetAP activity to provide a methionine salvage pathway, we evaluated the relationship between methionine metabolism and MetAP activity in Saccharomyces cerevisiae. We provide the first demonstration that yeast MetAP1 plays a significant role in methionine metabolism, namely, preventing premature activation of MET genes through MetAP function in methionine salvage. Interestingly, in cells lacking MetAP1, excess methionine dramatically inhibits cell growth. Growth inhibition is independent of the ability of methionine to repress MET genes and does not result from inhibition of synthesis of another metabolite, rather it results from product inhibition of MetAP2. Inhibition by methionine is selective for MetAP2 over MetAP1. These results provide an explanation for the previously observed dominance of MetAP1 in terms of N-terminal processing and cell growth in yeast. Additionally, differential regulation of the two isoforms may be indicative of different intracellular roles for the two enzymes.

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.