Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9.

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.

From transporter to transceptor: signaling from transporters provokes re-evaluation of complex trafficking and regulatory controls: endocytic internalization and intracellular trafficking of nutrient transceptors may, at least in part, be governed by their signaling function.

When cells are starved of their substrate, many nutrient transporters are induced. These undergo rapid endocytosis and redirection of their intracellular trafficking when their substrate becomes available again. The discovery that some of these transporters also act as receptors, or transceptors, suggests that at least part of the sophisticated controls governing the trafficking of these proteins has to do with their signaling function rather than with control of transport. In yeast, the general amino acid permease Gap1 mediates signaling to the protein kinase A pathway. Its endocytic internalization and intracellular trafficking are subject to amino acid control. Other nutrient transceptors controlling this signal transduction pathway appear to be subject to similar trafficking regulation. Transporters with complex regulatory control have also been suggested to function as transceptors in other organisms. Hence, precise regulation of intracellular trafficking in nutrient transporters may be related to the need for tight control of nutrient-induced signaling.

The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae.

PDK1 (phosphoinositide-dependent protein kinase 1) phosphorylates and activates PKA (cAMP-dependent protein kinase) in vitro. Docking of the HM (hydrophobic motif) in the C-terminal tail of the PKA catalytic subunits on to the PIF (PDK1-interacting fragment) pocket of PDK1 is a critical step in this activation process. However, PDK1 regulation of PKA in vivo remains controversial. Saccharomyces cerevisiae contains three PKA catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh [PKB (protein kinase B)-activating kinase homologue] protein kinases phosphorylate the activation loop of each Tpk in vivo with various efficiencies. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of PKA. Comparative analysis of the in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through an HM-PIF pocket interaction. Unlike Tpk1, mutagenesis of the activation loop site in Tpk2 does not abolish in vitro phosphorylation, suggesting that Tpk2 contains other, as yet uncharacterized, Pkh1 target sites. Tpk3 is poorly phosphorylated on its activation loop site, and this is due to the weak interaction of Tpk3 with Pkh1 because of the atypical HM found in Tpk3. In conclusion, the results of the present study show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.

Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9.

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.

The Hospital Recovery Scale: A clinically useful endpoint in patients hospitalized with influenza.

BACKGROUND: Currently, no single best primary endpoint exists for measuring the efficacy of treatments in seriously ill patients with respiratory infections, such as influenza, who require hospitalization. The Hospital Recovery Scale is an ordinal endpoint used to evaluate treatment outcomes in clinical studies of hospitalized patients infected with influenza. METHODS: To determine whether Hospital Recovery Scale outcomes correspond to those for other clinical endpoints in patients hospitalized due to influenza, data from the phase 3 randomized, double-blind ZORO clinical trial (NCT01231620) were analyzed. Randomized influenza-infected patients were divided into subgroups of interest based on prespecified baseline and infection-related characteristics, as well as randomized treatment arms (intravenous zanamivir 300 mg or 600 mg, or oral oseltamivir 75 mg). Clinical endpoints relevant to this population were included to analyze differences in outcomes between the subgroups, and correspondence of these endpoints and hospital recovery endpoint was evaluated. RESULTS: Data from 488 patients were analyzed. There were strong correlations (ρs > 0.8) between the Hospital Recovery Scale assessed on the day after completion of a 5-day antiviral therapy (Day 6) and both time to hospital discharge and time to intensive care unit discharge, and moderate to strong correlations (0.6 < ρs < 0.8) between the Hospital Recovery Scale on Day 6 and several other relevant clinical endpoints. CONCLUSIONS: The Hospital Recovery Scale is applicable as a primary endpoint in trials to evaluate new therapies for severely ill patients hospitalized due to influenza, and may have utility in other severe respiratory illnesses such as COVID-19.

Single versus Multiple Dose Ivermectin Regimen in Onchocerciasis-Infected Persons with Epilepsy Treated with Phenobarbital: A Randomized Clinical Trial in the Democratic Republic of Congo.

BACKGROUND: There is anecdotal evidence that ivermectin may decrease seizure frequency in Onchocerca volvulus-infected persons with epilepsy (PWE). METHODS: In October 2017, a 12-month clinical trial was initiated in rural Democratic Republic of Congo. PWE with onchocerciasis-associated epilepsy experiencing ≥2 seizures/month were randomly allocated to receive, over a one-year period, ivermectin once or thrice (group 1), while other onchocerciasis-infected PWE (OIPWE) were randomized to ivermectin twice or thrice (group 2). All participants also received anti-epileptic drugs. Data was analyzed using multiple logistic regression. RESULTS: We enrolled 197 participants. In an intent-to-treat analysis (data from group 1 and 2 combined), seizure freedom was more likely among OIPWE treated with ivermectin thrice (OR: 5.087, 95% CI: 1.378-19.749; p = 0.018) and twice (OR: 2.471, 95% CI: 0.944-6.769; p = 0.075) than in those treated once. Similarly, >50% seizure reduction was more likely among those treated with ivermectin twice (OR: 4.469, 95% CI: 1.250-16.620) and thrice (OR: 2.693, 95% CI: 1.077-6.998). Absence of microfilariae during the last 4 months increased the odds of seizure freedom (p = 0.027). CONCLUSIONS: Increasing the number of ivermectin treatments was found to suppress both microfilarial density and seizure frequency in OIPWE, suggesting that O. volvulus infection plays an etiological role in causing seizures.

Ivermectin as an adjuvant to anti-epileptic treatment in persons with onchocerciasis-associated epilepsy: A randomized proof-of-concept clinical trial.

INTRODUCTION: Recent findings from onchocerciasis-endemic foci uphold that increasing ivermectin coverage reduces the epilepsy incidence, and anecdotal evidence suggests seizure frequency reduction in persons with onchocerciasis-associated epilepsy, when treated with ivermectin. We conducted a randomized clinical trial to assess whether ivermectin treatment decreases seizure frequency. METHODS: A proof-of-concept randomized clinical trial was conducted in the Logo health zone in the Ituri province, Democratic Republic of Congo, to compare seizure frequencies in onchocerciasis-infected persons with epilepsy (PWE) randomized to one of two treatment arms: the anti-epileptic drug phenobarbital supplemented with ivermectin, versus phenobarbital alone. The primary endpoint was defined as the probability of being seizure-free at month 4. A secondary endpoint was defined as >50% reduction in seizure frequency at month 4, compared to baseline. Both endpoints were analyzed using multiple logistic regression. In longitudinal analysis, the probability of seizure freedom during the follow-up period was assessed for both treatment arms by fitting a logistic regression model using generalized estimating equations (GEE). RESULTS: Ninety PWE enrolled between October and November 2017 were eligible for analysis. A multiple logistic regression analysis showed a borderline association between ivermectin treatment and being seizure-free at month 4 (OR: 1.652, 95% CI 0.975-2.799; p = 0.062). There was no significant difference in the probability of experiencing >50% reduction of the seizure frequency at month 4 between the two treatment arms. Also, treatment with ivermectin did not significantly increase the odds of being seizure-free during the individual follow-up visits. CONCLUSION: Whether ivermectin has an added value in reducing the frequency of seizures in PWE treated with AED remains to be determined. A larger study in persons with OAE on a stable AED regimen and in persons with recent epilepsy onset should be considered to further investigate the potential beneficial effect of ivermectin treatment in persons with OAE. TRIAL REGISTRATION: Registration: www.clinicaltrials.gov; NCT03052998.

Novel mechanisms in nutrient activation of the yeast protein kinase A pathway.

In yeast the Protein Kinase A (PKA) pathway can be activated by a variety of nutrients. Fermentable sugars, like glucose and sucrose, trigger a spike in the cAMP level, followed by activation of PKA and phosphorylation of target proteins causing a.o. mobilization of reserve carbohydrates, repression of stress-related genes and induction of growth-related genes. Glucose and sucrose are sensed by a G-protein coupled receptor system that activates adenylate cyclase and also activates a bypass pathway causing direct activation of PKA. Addition of other essential nutrients, like nitrogen sources or phosphate, to glucose-repressed nitrogen- or phosphate-starved cells, also triggers rapid activation of the PKA pathway. In these cases cAMP is not involved as a second messenger. Amino acids are sensed by the Gap1 transceptor, previously considered only as an amino acid transporter. Recent results indicate that the amino acid ligand has to induce a specific conformational change for signaling. The same amino acid binding site is involved in transport and signaling. Similar results have been obtained for Pho84 which acts as a transceptor for phosphate activation of the PKA pathway. Ammonium activation of the PKA pathway in nitrogen-starved cells is mediated mainly by the Mep2 transceptor, which belongs to a different class of transporter proteins. Hence, different types of sensing systems are involved in control of the yeast PKA pathway by nutrients.