Impact of genetic variation on human CaMKK2 regulation by Ca2+ -calmodulin and multisite phosphorylation

The Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) is a key regulator of neuronal function and whole-body energy metabolism. Elevated CaMKK2 activity is strongly associated with prostate and hepatic cancers, whereas reduced CaMKK2 activity has been linked to schizophrenia and bipolar disease in humans. Here we report the functional effects of nine rare-variant point mutations that were detected in large-scale human genetic studies and cancer tissues, all of which occur close to two regulatory phosphorylation sites and the catalytic site on human CaMKK2. Four mutations (G87R, R139W, R142W and E268K) cause a marked decrease in Ca2+-independent autonomous activity, however S137L and P138S mutants displayed increased autonomous and Ca2+-CaM stimulated activities. Furthermore, the G87R mutant is defective in Thr85-autophosphorylation dependent autonomous activity, whereas the A329T mutation rendered CaMKK2 virtually insensitive to Ca2+-CaM stimulation. The G87R and R139W mutants behave as dominant-negative inhibitors of CaMKK2 signaling in cells as they block phosphorylation of the downstream substrate AMP-activated protein kinase (AMPK) in response to ionomycin. Our study provides insight into functionally disruptive, rare-variant mutations in human CaMKK2, which have the potential to influence risk and burden of disease associated with aberrant CaMKK2 activity in human populations carrying these variants

Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding

The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases

The autophagy initiator ULK1 sensitizes AMPK to allosteric drugs

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation

A critical comparison of integral projection and matrix projection models for demographic analysis

Structured demographic models are among the most common and useful tools in population biology. However, the introduction of integral projection models (IPMs) has caused a profound shift in the way many demographic models are conceptualized. Some researchers have argued that IPMs, by explicitly representing demographic processes as continuous functions of state variables such as size, are more statistically efficient, biologically realistic, and accurate than classic matrix projection models, calling into question the usefulness of the many studies based on matrix models. Here, we evaluate how IPMs and matrix models differ, as well as the extent to which these differences matter for estimation of key model outputs, including population growth rates, sensitivity patterns, and life spans. First, we detail the steps in constructing and using each type of model. Second, we present a review of published demographic models, concentrating on size-based studies, which shows significant overlap in the way IPMs and matrix models are constructed and analyzed. Third, to assess the impact of various modeling decisions on demographic predictions, we ran a series of simulations based on size-based demographic data sets for five biologically diverse species. We found little evidence that discrete vital rate estimation is less accurate than continuous functions across a wide range of sample sizes or size classes (equivalently bin numbers or mesh points). Most model outputs quickly converged with modest class numbers (≥10), regardless of most other modeling decisions. Another surprising result was that the most commonly used method to discretize growth rates for IPM analyses can introduce substantial error into model outputs. Finally, we show that empirical sample sizes generally matter more than modeling approach for the accuracy of demographic outputs. Based on these results, we provide specific recommendations to those constructing and evaluating structured population models. Both our literature review and simulations question the treatment of IPMs as a clearly distinct modeling approach or one that is inherently more accurate than classic matrix models. Importantly, this suggests that matrix models, representing the vast majority of past demographic analyses available for comparative and conservation work, continue to be useful and important sources of demographic information.Support for this work was provided by NSF awards 1146489, 1242558, 1242355, 1353781, 1340024, 1753980, and 1753954, 1144807, 0841423, and 1144083. Support also came from USDA NIFA Postdoctoral Fellowship (award no. 2019-67012-29726/project accession no. 1019364) for R. K. Shriver; the Swiss Polar Institute of Food and Agriculture for N. I. Chardon; the ICREA under the ICREA Academia Programme for C. Linares; and SERDP contract RC-2512 and USDA National Institute of Food and Agriculture, Hatch project 1016746 for A .M. Louthan. This is Contribution no. 21-177-J from the Kansas Agricultural Experiment Station

Antimicrobial treatment practices among Ugandan children with suspicion of central nervous system infection.

Acute central nervous system (CNS) infections in children in sub-Saharan Africa are often fatal. Potential contributors include late presentation, limited diagnostic capacity and inadequate treatment. A more nuanced understanding of treatment practices with a goal of optimizing such practices is critical to prevent avoidable case fatality. We describe empiric antimicrobial treatment, antibiotic resistance and treatment adequacy in a prospective cohort of 459 children aged two months to 12 years hospitalised for suspected acute CNS infections in Mbarara, Uganda, from 2009 to 2012. Among these 459 children, 155 had a laboratory-confirmed diagnosis of malaria (case-fatality rate [CFR] 14%), 58 had bacterial infections (CFR 24%) and 6 children had mixed malaria and bacterial infections (CFR 17%). Overall case fatality was 18.1% (n = 83). Of 219 children with laboratory-confirmed malaria and/or bacterial infections, 182 (83.1%) received an adequate antimalarial and/or antibiotic on the day of admission and 211 (96.3%) within 48 hours of admission. The proportion of those receiving adequate treatment was similar among survivors and non-survivors. All bacterial isolates were sensitive to ceftriaxone except one Escherichia coli isolate with extended-spectrum beta-lactamase (ESBL). The observed high mortality was not a result of inadequate initial antimicrobial treatment at the hospital. The epidemiology of CNS infection in this setting justifies empirical use of a third-generation cephalosporin, however antibiotic resistance should be monitored closely

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body

Infant and young child nutritional status and their caregivers' feeding knowledge and hygiene practices in internally displaced person camps, Somalia

Background: In an attempt to design an educational programme targeting caregivers of children aged 6 to 59 months in internally displaced persons camps in Somalia, the objective of this study was twofold. First, to explore the nutritional situation of all children aged 6-59 months enrolled in a nutrition programme provided by Save the Children in 2017 in internally displaced persons camps. Second, to identify gaps in the caregivers' hygiene and feeding practices. Methods: In a study of 1655 households, 1655 caregivers for 2370 children aged 6 to 59 months enrolled in a nutrition programme provided by Save the Children answered an adapted questionnaire on hygiene and feeding practices. At the same time, based on standard criteria in the questionnaire, naturalistic observations of caregivers’ hygiene practices were conducted. Every child in the study was measured with anthropometric Mid-Upper-Arm Circumference measurements for the classification of Moderate Acute Malnutrition, Severe Acute Malnutrition and Global Acute Malnutrition. Descriptive statistics were used for analysis. Results: 1) There was Severe (12.1%) and Global Acute (19.9%) Malnutrition among children included in the nutrition programme, more frequently in the 6-24 month age group compared to the 25-59 month age group (p<0.01). 2). The practices in the households were below what could generally be considered hygienic. 3) There was poor caregivers’ knowledge of breastfeeding benefits and complementary foods. Conclusion: Child malnutrition might derive from gaps in the caregiver's knowledge, attitudes, and practices regarding hygiene and infant feeding. An awareness of these gaps can be helpful in designing future educational programmes that target caregivers, particularly in at-risk population groups

AMP-activated protein kinase - not just an energy sensor

Orthologues of AMP-activated protein kinase (AMPK) occur in essentially all eukaryotes as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits. The canonical role of AMPK is as an energy sensor, monitoring levels of the nucleotides AMP, ADP, and ATP that bind competitively to the γ subunit. Once activated, AMPK acts to restore energy homeostasis by switching on alternate ATP-generating catabolic pathways while switching off ATP-consuming anabolic pathways. However, its ancestral role in unicellular eukaryotes may have been in sensing of glucose rather than energy. In this article, we discuss a few interesting recent developments in the AMPK field. Firstly, we review recent findings on the canonical pathway by which AMPK is regulated by adenine nucleotides. Secondly, AMPK is now known to be activated in mammalian cells by glucose starvation by a mechanism that occurs in the absence of changes in adenine nucleotides, involving the formation of complexes with Axin and LKB1 on the surface of the lysosome. Thirdly, in addition to containing the nucleotide-binding sites on the γ subunits, AMPK heterotrimers contain a site for binding of allosteric activators termed the allosteric drug and metabolite (ADaM) site. A large number of synthetic activators, some of which show promise as hypoglycaemic agents in pre-clinical studies, have now been shown to bind there. Fourthly, some kinase inhibitors paradoxically activate AMPK, including one (SU6656) that binds in the catalytic site. Finally, although downstream targets originally identified for AMPK were mainly concerned with metabolism, recently identified targets have roles in such diverse areas as mitochondrial fission, integrity of epithelial cell layers, and angiogenesis

The X-Ray Crystal Structure of Escherichia coli Succinic Semialdehyde Dehydrogenase; Structural Insights into NADP+/Enzyme Interactions

In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and gamma-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease