Kristie Grove Bridges

Detection and purification of tyrosine-sulfated proteins using a novel anti-sulfotyrosine monoclonal antibody

Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2-/- mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2-/- males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms.

Salivary uric acid as a noninvasive biomarker of metabolic syndrome

BackgroundElevated serum uric acid is associated with obesity, hypertension and metabolic syndrome. Because a linear relationship exists between serum and salivary uric acid (SUA) concentration, saliva testing may be a useful noninvasive approach for monitoring cardiometabolic risk. The goal of this pilot study was to determine if SUA is increased in patients with metabolic syndrome and to investigate correlations between SUA and individual cardiometabolic risk factors.FindingsVolunteers between the ages of 18 and 65 without conditions known to affect serum uric acid levels were recruited. Height, weight, blood pressure and waist circumference were measured and a full lipid panel along with fasting blood glucose was obtained. Saliva samples were collected and uric acid levels were determined. 78 volunteers, 35% of whom had metabolic syndrome, completed the study. SUA was significantly elevated in patients with metabolic syndrome (p=.002). The incidence of metabolic syndrome in the 4th quartile for SUA was 67% compared to 25% in quartiles1-3 combined. Significant correlations were seen between SUA and systolic blood pressure (r=.440, p=.000), diastolic blood pressure ( r=.304, p=.007), waist circumference (r=.332, p=.003), BMI ( r=.269, p=.018), fasting blood glucose ( r=.341, p=.002), triglycerides (r=.410, p=.000), HDL ( r=.237, p=.036) and the number of cardiometabolic risk factors present (r=0.257, p=.023).ConclusionsThese results suggest that SUA may be a useful biomarker for noninvasive monitoring of cardiometabolic risk. Larger studies are needed to validate this approach.

Detection of homocysteine and C-reactive protein in the saliva of healthy adults: comparison with blood levels.

Inflammation and cardiovascular disease are associated with elevated serum levels of C-Reactive Protein (CRP) and homocysteine. The presence of both molecules in saliva provides an opportunity for development of non-invasive assessments of disease risk. However, salivary CRP and homocysteine reference ranges and their correlation with serum levels are unknown. This study investigated if CRP and homocysteine could be routinely detected in the saliva of healthy adults and the relationship between salivary and blood levels. CRP and homocysteine concentrations were determined using ELISA and enzymatic assays respectively. Homocysteine was detected in only two saliva samples (n = 55). CRP was measurable in all saliva samples (range: 0.05 to 64.3 μg/L; median =1.2 μg/L) and plasma samples (range: 0.14 to 31.1 mg/L; median = 2.0 mg/L). Regression analysis demonstrated no relationship between CRP concentration in saliva and plasma (R 2 = 0.001). Generalized linear models including variables such as saliva flow rate and time since eating or drinking also did not pass lack of fit testing. Therefore, a relationship between CRP concentration in saliva and blood could not be established in this group of subjects. More sensitive detection methods are needed to determine if a correlation between salivary and serum homocysteine levels exists.

Identification of Crucial Hydrogen-Bonding Residues for the Interaction of Herpes Simplex Virus DNA Polymerase Subunits via Peptide Display, Mutational, and Calorimetric Approaches

ABSTRACT The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 μM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial α-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional 1H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal α-helices separated by a less ordered region. A series of “alanine scan” peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.

Use of the triglyceride to HDL cholesterol ratio for assessing insulin sensitivity in overweight and obese children in rural Appalachia.

Abstract Background: Studies have suggested that triglyceride to HDL-cholesterol ratio (TRG/HDL) is a surrogate marker of insulin resistance (IR), but information regarding its use in pediatric patients is limited. This study investigated the ability of TRG/HDL ratio to assess IR in obese and overweight children. Methods: The sample consisted of de-identified electronic medical records of patients aged 10–17 years (n=223). Logistic regression was performed using TRG/HDL ratio as a predictor of hyperinsulinemia or IR defined using homeostasis model assessment score. Results: TRG/HDL ratio had limited ability to predict hyperinsulinemia (AUROC 0.71) or IR (AUROC 0.72). Although females had higher insulin levels, male patients were significantly more likely to have hypertriglyceridemia and impaired fasting glucose. Conclusions: TRG/HDL ratio was not adequate for predicting IR in this population. Gender differences in the development of obesity-related metabolic abnormalities may impact the choice of screening studies in pediatric patients.

Inappropriate Use of Homeostasis Model Assessment Cutoff Values for Diagnosing Insulin Resistance in Pediatric Studies

Background Assessing pediatric patients for insulin resistance is one way to identify those who are at a high risk of developing type 2 diabetes mellitus. The homoeostasis model assessment (HOMA) is a measure of insulin resistance based on fasting blood glucose and insulin levels. Although this measure is widely used in research, cutoff values for pediatric populations have not been established. Objective To assess the validity of HOMA cutoff values used in pediatric studies published in peer-reviewed journals. Methods Studies published from January 2010 to December 2015 were identified through MEDLINE. Initial screening of abstracts was done to select studies that were conducted in pediatric populations and used HOMA to assess insulin resistance. Subsequent full-text review narrowed the list to only those studies that used a specific HOMA score to diagnose insulin resistance. Each study was classified as using a predetermined fixed HOMA cutoff value or a cutoff that was a percentile specific to that population. For studies that used a predetermined cutoff value, the references cited to provide evidence in support of that cutoff were evaluated. Results In the 298 articles analyzed, 51 different HOMA cutoff values were used to classify patients as having insulin resistance. Two hundred fifty-five studies (85.6%) used a predetermined fixed cutoff value, but only 72 (28.2%) of those studies provided a reference that supported its use. One hundred ten studies (43%) that used a fixed cutoff either cited a study that did not mention HOMA or provided no reference at all. Tracing of citation history indicated that the most commonly used cutoff values were ultimately based on studies that did not validate their use for defining insulin resistance. Conclusion Little evidence exists to support HOMA cutoff values commonly used to define insulin resistance in pediatric studies. These findings highlight the importance of validating study design elements when training medical students and novice investigators. Using available data to generate population ranges for HOMA would improve its clinical utility.