Targeting Rab-RILPL interactions as a strategy to downregulate pathogenic LRRK2 in Parkinson's disease.

Familial Parkinson's disease (PD) is frequently linked to multiple disease-causing mutations within Leucine-Rich Repeat Protein Kinase 2 (LRRK2), leading to aberrant kinase activity. Multiple pathogenic effects of enhanced LRRK2 activity have been identified, including loss of cilia and centrosomal cohesion defects. When phosphorylated by LRRK2, Rab8a and Rab10 bind to phospho-specific RILPL effector proteins. RILPL-mediated accumulation of pRabs proximal to the mother centriole is critical for initiating deficits in ciliogenesis and centrosome cohesion mediated by LRRK2. We hypothesized that Rab-derived phospho-mimics may serve to block phosphorylated Rab proteins from docking with RILPL in the context of hyperactive LRRK2 mutants. This would serve as an alternative strategy to downregulate pathogenic signaling mediated by LRRK2, rather than targeting LRRK2 kinase activity itself. To test this theory, we designed a series of constrained peptides mimicking phosphorylated Switch II derived from Rab8. These RILPL interacting peptides, termed RIP, were further shown to permeate cells. Further, several peptides were found to bind RILPL2 and restore ciliogenesis and centrosomal cohesion defects in cells expressing PD-associated mutant LRRK2. This research demonstrates the utility of constrained peptides as downstream inhibitors to target pathogenic LRRK2 activity and may provide an alternative approach to target specific pathways activated by LRRK2.

Beyond the Food Systems Summit: Linking Recommendations to Action-The True Cost of Food.

A transformation of food systems is needed to achieve the 17 Sustainable Development Goals specified in the 2030 Agenda for Sustainable Development. Recognizing the true costs and benefits of food production and consumption can help guide public policy decisions to effectively transform food systems in support of sustainable healthy diets. A new, expanded framework is presented that allows the quantification of costs and benefits in three domains: health, environmental, and social. The implications for policy makers are discussed. Curr Dev Nutr 2023;x:xx.

Doubly Constrained C-terminal of Roc (COR) Domain-Derived Peptides Inhibit Leucine-Rich Repeat Kinase 2 (LRRK2) Dimerization.

Missense mutations along the leucine-rich repeat kinase 2 (LRRK2) protein are a major contributor to Parkinson's Disease (PD), the second most commonly occurring neurodegenerative disorder worldwide. We recently reported the development of allosteric constrained peptide inhibitors that target and downregulate LRRK2 activity through disruption of LRRK2 dimerization. In this study, we designed doubly constrained peptides with the objective of inhibiting C-terminal of Roc (COR)-COR mediated dimerization at the LRRK2 dimer interface. We show that the doubly constrained peptides are cell-permeant, bind wild-type and pathogenic LRRK2, inhibit LRRK2 dimerization and kinase activity, and inhibit LRRK2-mediated neuronal apoptosis, and in contrast to ATP-competitive LRRK2 kinase inhibitors, they do not induce the mislocalization of LRRK2 to skein-like structures in cells. This work highlights the significance of COR-mediated dimerization in LRRK2 activity while also highlighting the use of doubly constrained peptides to stabilize discrete secondary structural folds within a peptide sequence.

Targeting the WASF3 complex to suppress metastasis.

Wiskott-Aldrich syndrome protein family members (WASF) regulate the dynamics of the actin cytoskeleton, which plays an instrumental role in cancer metastasis and invasion. WASF1/2/3 forms a hetero-pentameric complex with CYFIP1/2, NCKAP1/1 L, Abi1/2/3 and BRK1 called the WASF Regulatory Complex (WRC), which cooperatively regulates actin nucleation by WASF1/2/3. Activation of the WRC enables actin networking and provides the mechanical force required for the formation of lamellipodia and invadopodia. Although the WRC drives cell motility essential for several routine physiological functions, its aberrant deployment is observed in cancer metastasis and invasion. WASF3 expression is correlated with metastatic potential in several cancers and inversely correlates with overall progression-free survival. Therefore, disruption of the WRC may serve as a novel strategy for targeting metastasis. Given the complexity involved in the formation of the WRC which is largely comprised of large protein-protein interfaces, there are currently no inhibitors for WASF3. However, several constrained peptide mimics of the various protein-protein interaction interfaces within the WRC were found to successfully disrupt WASF3-mediated migration and invasion. This review explores the role of the WASF3 WRC in driving metastasis and how it may be selectively targeted for suppression of metastasis.

In Silico Optimized Stapled Peptides Targeting WASF3 in Breast Cancer.

Wiskott-Aldrich Syndrome Protein Family (WASF) members regulate actin cytoskeletal dynamics, and WASF3 is directly associated with breast cancer metastasis and invasion. WASF3 forms a heteropentameric complex with CYFIP, NCKAP, ABI, and BRK1, called the WASF Regulatory Complex (WRC), which cooperatively regulates actin nucleation by WASF3. Since aberrant deployment of the WRC is observed in cancer metastasis and invasion, its disruption provides a novel avenue for targeting motility in breast cancer cells. Here, we report the development of a second generation WASF3 mimetic peptide, WAHMIS-2, which was designed using a combination of structure-guided design, homology modeling, and in silico optimization to disrupt binding of WASF3 to the WRC. WAHMIS-2 was found to permeate cells and inhibit cell motility, invasion, and MMP9 expression with greater potency than its predecessor, WAHM1. Targeted disruption of WASF3 from the WRC may serve as a useful strategy for suppression of breast cancer metastasis.

The Role of Household Assets in Improving Women's Dietary Diversity in Ethiopia.

Background: ENGINE (Empowering New Generations for Improved Nutrition and Economic Opportunities) was a US Agency for International Development (USAID)-funded project implemented in Ethiopia from 2011 to 2016. ENGINE used a multisectoral approach to achieve the goals articulated in the Government of Ethiopia's National Nutrition Plan, among which is improvement in the dietary intakes of women and preschool-aged children. Objectives: The objectives of the present research are 2-fold: 1) to document trends in women's dietary diversity (WDD) and 2) to identify factors associated with dietary diversity for women. Methods: Descriptive statistics and multivariate, pooled analyses were calculated. Results: Results indicate that WDD was low, ranging, on average, from 3.0 to 4.0 (out of a possible 10). Across the time points covered reflecting pregnancy and 1-y postpartum, only ∼13% to 17% of women met the Minimum Dietary Diversity Score for Women (MDD-W). Conclusions: The production of both food and cash crops and the rearing of livestock were significant predictors of improved dietary diversity in women. The focus of ENGINE on a diverse set of agricultural activities improved WDD and MDD-W in a population of women where dietary diversity is poor.

Dietary Outcomes, Nutritional Status, and Household Water, Sanitation, and Hygiene (WASH) Practices.

Background: The Government of Ethiopia has made a major commitment toward improving food security, diet, nutrition, and health through a series of national nutrition plans. The focus of these plans is on providing both nutrition-specific as well as nutrition-sensitive approaches for achieving national priorities for health and nutrition. The present study conducted a secondary analysis of data provided through a larger birth cohort study conducted in Ethiopia between 2014 and 2016. Objectives: The overall objectives of this research were to assess the relation between minimum dietary diversity in women and water, sanitation, and hygiene (WASH), and evaluate the association between midupper arm circumference (MUAC) in women and WASH. Methods: In addition to descriptive statistics, the study used mixed effects logistic regression analyses to investigate the relation between dietary diversity, MUAC, and household WASH practices. Results: Improved WASH practices were associated with an increased probability (p = 0.04) that a woman would consume a diet with foods from 5 or more food groups. A beneficial effect was observed for improved WASH practices and a decrease in low MUAC. Improved household WASH practices were successful in contributing to improved dietary diversity in women as well as an improved MUAC. Conclusions: Interventions aimed at improving the diet and nutritional status of women during and after pregnancy should include relevant WASH components as essential elements in multisector nutrition programming.

Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes.

Generation of the prototypic second messenger cAMP instigates numerous signaling events. A major intracellular target of cAMP is Protein kinase A (PKA), a Ser/Thr protein kinase. Where and when this enzyme is activated inside the cell has profound implications on the functional impact of PKA. It is now well established that PKA signaling is focused locally into subcellular signaling "islands" or "signalosomes." The A-Kinase Anchoring Proteins (AKAPs) play a critical role in this process by dictating spatial and temporal aspects of PKA action. Genetically encoded biosensors, small molecule and peptide-based disruptors of PKA signaling are valuable tools for rigorous investigation of local PKA action at the biochemical level. This chapter focuses on approaches to evaluate PKA signaling islands, including a simple assay for monitoring the interaction of an AKAP with a tunable PKA holoenzyme. The latter approach evaluates the composition of PKA holoenzymes, in which regulatory subunits and catalytic subunits can be visualized in the presence of test compounds and small-molecule inhibitors.

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

Household Decision-Making: Implications for Production, Consumption, and Sale of Nutrient-Rich Foods.

Household decision-making influences choices related to the production, sale, purchase, and consumption of nutrient-rich foods. The present study assessed the effect of household decision-making in 2 regions of Ethiopia within 2 groups of households, most vulnerable households and model farmer households. The study focused on identifying barriers and facilitators relating to decisions about nutrient-rich foods-in this case fruits, vegetables, and animal source foods. The results provide insights into how future agricultural programs can affect key aspects of decision-making to maximize the positive impacts on diet and food security.

Allosteric Inhibition of Parkinson's-Linked LRRK2 by Constrained Peptides.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein with dual kinase and GTPase function that is commonly mutated in both familial and idiopathic Parkinson's Disease (PD). While dimerization of LRRK2 is commonly detected in PD models, it remains unclear whether inhibition of dimerization can regulate catalytic activity and pathogenesis. Here, we show constrained peptides that are cell-penetrant, bind LRRK2, and inhibit LRRK2 activation by downregulating dimerization. We further show that inhibited dimerization decreases kinase activity and inhibits ROS production and PD-linked apoptosis in primary cortical neurons. While many ATP-competitive LRRK2 inhibitors induce toxicity and mislocalization of the protein in cells, these constrained peptides were found to not affect LRRK2 localization. The ability of these peptides to inhibit pathogenic LRRK2 kinase activity suggests that disruption of dimerization may serve as a new allosteric strategy to downregulate PD-related signaling pathways.

Targeted disruption of PKC from AKAP signaling complexes.

Protein Kinase C (PKC) is a member of the AGC subfamily of kinases and regulates a wide array of signaling pathways and physiological processes. Protein-protein interactions involving PKC and its scaffolding partners dictate the spatiotemporal dynamics of PKC activity, including its access to activating second messenger molecules and potential substrates. While the A Kinase Anchoring Protein (AKAP) family of scaffold proteins universally bind PKA, several were also found to scaffold PKC, thereby serving to tune its catalytic output. Targeting these scaffolding interactions can further shed light on the effect of subcellular compartmentalization on PKC signaling. Here we report the development of two hydrocarbon stapled peptides, CSTAD5 and CSTAD6, that are cell permeable and bind PKC to disrupt PKC-gravin complex formation in cells. Both constrained peptides downregulate PMA-induced cytoskeletal remodeling that is mediated by the PKC-gravin complex as measured by cell rounding. Further, these peptides downregulate PKC substrate phosphorylation and cell motility. To the best of our knowledge, no PKC-selective AKAP disruptors have previously been reported and thus CSTAD5 and CSTAD6 are novel disruptors of PKC scaffolding by AKAPs and may serve as powerful tools for dissecting AKAP-localized PKC signaling.

Social and behaviour change communication to improve child feeding practices in Ethiopia.

Social and behaviour change communication (SBCC) interventions can positively affect optimal nutritional practices. This study evaluated the added value of a virtual facilitator tool to an enhanced community conversation (ECC) programme to improve infant and young child feeding (IYCF) practice among children under the Growth through Nutrition Activity programme in Ethiopia. The study used a quasi-experimental design with a control group. Pregnant and/or lactating women were the study population for both study groups. The intervention (ECC + VF) group received all the same components as the control group but had the addition of in-person ECC meetings supplemented with audio-recorded virtual facilitators (VF) sessions designed to complement the monthly meeting lesson or topic. A difference in difference analysis was employed using generalized linear mixed model (GLMM) in Stata version 15.0 (Stata Corporation, College Station, TX). A p-value of less than or equal to 0.05 was considered significant for all tests. Accordingly, a 13.6% change in iron folic acid (IFA) intake for 3 months and above was observed in the intervention group. Even though not statistically significant, large to moderate positive changes in child minimum diet diversity (20%), minimum acceptable diet (18%) and women diet diversity (7.9%) were observed in the intervention group. This study identified the use of virtual facilitators as a modality to transmit standard nutrition messages during ECC programmes for optimal IYCF practices. The findings strengthen the notion that using a combination of SBCC approaches has advantage over a single method in improving important nutritional practices.

cAMP-Dependent Signaling Pathways as Potential Targets for Inhibition of Plasmodium falciparum Blood Stages.

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites.

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCßII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

Conversion of soluble recalcitrant phosphorus to recoverable orthophosphate form using UV/H2O2.

Soluble non-reactive phosphorus (sNRP), such as inorganic polyphosphates and organic P, is not effectively removed by conventional physicochemical processes. This can impede water resource reclamation facilities' ability to meet stringent total P regulations. This study investigated a UV/H2O2 advanced oxidation process (AOP) for converting sNRP to the more readily removable/recoverable soluble reactive P (sRP), or orthophosphate, form. Synthetic water spiked with four sNRP compounds (beta-glycerol phosphate, phytic acid, triphosphate, and hexa-meta phosphate) at varying H2O2 concentration, UV fluence, pH, and temperature was initially tested. These compounds represent simple, complex, organic, and inorganic forms of sNRP potentially found in wastewater. The efficiency of sNRP to sRP conversion depended on whether the sNRP compound was organic or inorganic and the complexity of its chemical structure. Using 1 mM H2O2 and 0.43 J/cm2 (pH 7.5, 22 °C), conversion of the simple organic beta-glycerol phosphate to sRP was 38.1 ± 2.9%, which significantly exceeded the conversion of the other sNRP compounds. Although conversion was achieved, the electrical energy per order (EEO) was very high at 5.2 × 103 ± 5.2 × 102 kWh/m3. Actual municipal wastewater secondary effluent, with sNRP accounting for 15% of total P, was also treated using UV/H2O2. No wastewater sNRP to sRP conversion was observed, ostensibly due to interference from wastewater constituents. Wastewater utilities that have difficulty meeting stringent P levels might be able to target simple organic sNRP compounds, though alternative processes beyond UV/H2O2 need to be explored to overcome interference from wastewater constituents and target more complex organic and inorganic sNRP compounds.

Essential Health and Nutrition Service Provision during the COVID-19 Pandemic: Lessons from Select Ethiopian Woredas.

The world has faced a public health emergency due to the emergence of the COVID-19 pandemic. A cross-sectional study with mixed methods was conducted to review the status of maternal and child health care and nutrition service delivery during the early months of the pandemic in woredas (districts) targeted by the Growth through Nutrition Activity, a multi-sectoral nutrition project in Ethiopia. Comparison with the previous year showed some decline in key maternal and child health and nutrition services, with more pronounced effects during the early months of March and April before coordinated effort and standard guidance were well established. A recovery of most services was likely due in no small part to a range of mitigation interventions implemented by respective health workers and institutions, supervising government organizations, and through support from non-governmental organizations.

Kinetic Characterization of ASXL1/2-Mediated Allosteric Regulation of the BAP1 Deubiquitinase.

BAP1 is an ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like protein ASXL2. Cancer-related BAP1 mutations/deletions lead to loss-of-function by targeting the catalytic ubiquitin C-terminal hydrolase (UCH) or UCH37-like domain (ULD) domains of BAP1, and the latter disrupts binding to ASXL2, an obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of domains involved in forming the enzymatically active complex are unknown. Here, we report the molecular dynamics, kinetics, and stoichiometry of these interactions. We demonstrate that interactions between BAP1 and ASXL2 are direct, specific, and stable to biochemical and biophysical manipulations as detected by isothermal titration calorimetry (ITC), GST association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 activity. A stable ternary complex is formed, comprised of the BAP1-UCH, BAP1-ULD, and ASXL2-AB domains. Stoichiometric analysis revealed that one molecule of the ULD domain directly interacts with one molecule of the AB box. Real-time kinetic analysis of the ULD/AB protein complex to the BAP1-UCH domain, based on surface plasmon resonance, indicated that formation of the ULD/AB complex with the UCH domain is a single-step event with fast association and slow dissociation rates. In vitro experiments validated in cells that the ASXL-AB box directly regulates BAP1 activity. IMPLICATIONS: Collectively, these data elucidate molecular interactions between specific protein domains regulating BAP1 deubiquitinase activity, thus establishing a foundation for small-molecule approaches to reactivate latent wild-type BAP1 catalytic activity in BAP1-mutant cancers.

Transforming Food Systems: The Missing Pieces Needed to Make Them Work.

Food systems lie squarely at the intersection of several overarching goals of the UN and member states, as embodied in the Sustainable Development Goals, including eliminating poverty, hunger, and malnutrition in all its forms, achieving good health and well-being, while promoting environmental sustainability. The need for radical transformation of current food systems is inescapable if the world is to achieve one, let alone all, of these goals. Meeting this challenge will inevitably be disruptive to current food systems, carry costs, and be politically onerous. But the projected benefits far outweigh these difficulties. This commentary spells out the complexity of issues that need to be tackled to design and implement food systems that improve diets, nutrition, and health in an equitable fashion, while simultaneously respecting planetary boundaries. Six critical domains are identified that must be addressed for the successful transformation of food systems: 1) reinvent agriculture, 2) transform food environments for healthy diets, 3) mitigate climate change, 4) productively engage the private sector, 5) influence public policy priorities, and 6) establish true cost accounting of food. Because science is crucial for each of these domains, a research-driven strategy, emphasizing a collaborative process, is outlined. Bold, new, but technically and politically feasible actions are needed to effectively transform current food systems.

Nutrition Policy and Governance in Ethiopia: What Difference Does 5 Years Make?

Two studies, ENGINE (Empowering New Generations to Improve Nutrition and Economic opportunities) and Growth through Nutrition, were conducted in the same 4 regions of Ethiopia approximately 5 years apart. A similar protocol using a quantitative and qualitative survey of key informants at the subnational level was used to explore barriers and facilitators for implementation of the country's national multisector nutrition plan. Noticeable differences were observed, including a change in the perception of the nutrition problems in pregnant women and preschool aged children and greater awareness of the multisector plan. Poor coordination and collaboration were still noted in both time periods. A key issue highlighted was the need to keep up the momentum for multisector approaches to improve nutrition in the policy agenda.