Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G6P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G6P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. Based on these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme, but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation

Ohio Economic Insects and Related Anthropods

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Structural insights into the inhibited state of Glycogen Synthase

poster abstractGlycogen is an osmotically inert polymer of glucose, synthesized during times of nutritional sufficiency so that it can be rapidly catabolized when there is an energy demand1. Glycogen synthase (GS) is responsible for the bulk of its synthesis by transferring glucose from UDPG to an existing glucose polymer1. Eukaryotic GS is allosterically activated by glucose-6-phosphate (G6P) and negatively regulated by covalent phosphorylation2. A cluster of six arginine residues are conserved across all eukaryotic species which determine the enzyme’s ability to respond to these activating and inhibitory signals2. Prior structural studies from our lab had shed light on the dephosphorylated and activated state of the enzyme3. However, little is known on the phosphorylated state of the enzyme. For structural studies on the inhibited state, we used the yGsy2R589/592A mutant as a surrogate since it has a basal activity state similar to the inhibited phosphorylated state. We solved the structure of the mutant to a resolution of 3.3 Å. While the overall structural arrangement of the tetramer is similar to the basal state enzyme, the interfaces are more closed. In particular, the N-terminal Rossmann-fold domain is rotated toward the interface by 5.9°, limiting access to the active site by the acceptor end of the glycogen chain. Coincident with this domain closure, we also observed that the the distance between the regulatory helices of adjacent monomers are moved closer to one another. Based on this observation, we hypothesized we could develop a reversible redox regulatory feature in the enzyme by substituting cysteine residues for arginines 581 and 592, which lie across from each other at the interface. Consistent with our hypothesis, the yGsy2R581/592C double mutant exhibited very low activity, and could not be activated by G6P. However, normal function of the enzyme could be restored in the presence of reducing agents like DTT, BME and TCEP. Taken together, our mutational work demonstrates that the conserved arginine cluster in the regulatory helix, both regulates the enzyme’s response to signaling inputs and keeps the enzyme in a basal state conformation that is poised to respond to the activating and inhibitory inputs

Psychometric Evaluation and Design of Patient-Centered Communication Measures for Cancer Care Settings

Objective To evaluate the psychometric properties of questions that assess patient perceptions of patient-provider communication and design measures of patient-centered communication (PCC). Methods Participants (adults with colon or rectal cancer living in North Carolina) completed a survey at 2 to 3 months post-diagnosis. The survey included 87 questions in six PCC Functions: Exchanging Information, Fostering Health Relationships, Making Decisions, Responding to Emotions, Enabling Patient Self-Management, and Managing Uncertainty. For each Function we conducted factor analyses, item response theory modeling, and tests for differential item functioning, and assessed reliability and construct validity. Results Participants included 501 respondents; 46% had a high school education or less. Reliability within each Function ranged from 0.90 to 0.96. The PCC-Ca-36 (36-question survey; reliability=0.94) and PCC-Ca-6 (6-question survey; reliability=0.92) measures differentiated between individuals with poor and good health (i.e., known-groups validity) and were highly correlated with the HINTS communication scale (i.e., convergent validity). Conclusion This study provides theory-grounded PCC measures found to be reliable and valid in colorectal cancer patients in North Carolina. Future work should evaluate measure validity over time and in other cancer populations. Practice implications The PCC-Ca-36 and PCC-Ca-6 measures may be used for surveillance, intervention research, and quality improvement initiatives

Rhythm in the speech of a person with right hemisphere damage: Applying the pairwise variability index

Although several aspects of prosody have been studied in speakers with right hemisphere damage (RHD), rhythm remains largely uninvestigated. This study compares the rhythm of an Australian English speaker with right hemisphere damage (due to a stroke, but with no concomitant dysarthria) to that of a neurologically unimpaired individual. The speakers' rhythm is compared using the pairwise variability index (PVI) which allows for an acoustic characterization of rhythm by comparing the duration of successive vocalic and intervocalic intervals. A sample of speech from a structured interview between a speech and language therapist and each participant was analysed. Previous research has shown that speakers with RHD may have difficulties with intonation production, and therefore it was hypothesized that there may also be rhythmic disturbance. Results show that the neurologically normal control uses a similar rhythm to that reported for British English (there are no previous studies available for Australian English), whilst the speaker with RHD produces speech with a less strongly stress-timed rhythm. This finding was statistically significant for the intervocalic intervals measured (t(8) = 4.7, p < .01), and suggests that some aspects of prosody may be right lateralized for this speaker. The findings are discussed in relation to previous findings of dysprosody in RHD populations, and in relation to syllable-timed speech of people with other neurological conditions